Patent Application
20210247408
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 8, 2019, is named SequenceListing_ST25.txt and is 19,727 bytes in size.
The invention relates to diagnosis, prevention, and treatment of diseases and conditions associated with the functions of prion-like or Tetz-proteins.
Prions are molecules characterized by self-propagation, which can undergo a conformational switch leading to the creation of new prions. Prion proteins have originally been associated with the development of mammalian pathologies; however, recently they have been shown to contribute to the environmental adaptation in a variety of prokaryotic and eukaryotic organisms. Prions lead to the misfolding of proteins. Together with the previously demonstrated pathogenic roles of prions during the development of different mammalian diseases, including neurodegenerative disease, prions have recently been shown to represent an important functional component in many prokaryotic and eukaryotic organisms and bacteriophages.
It is known that there are prion proteins capable of acquiring a specific molecule configuration denoted as beta-structure. Prions with a beta structure have special physicochemical and functional biological characteristics and possess thermal stability. Prions play a role in the emergence of various human and animal diseases. Prion diseases are characterized by one or more symptoms of dementia and/or cognitive impairments, that include, for example, Creutzfeldt-Jakob Disease, variant Creuzfeldt-Jakob Disease, Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia, and kuru. Simultaneously, there are thermostable proteins that do not have prion-like sequences of amino acids in their structure. Some thermostable proteins are formed under external effects, including proteases. The unique characteristics of prions allow them to actively participate in changing the properties of other proteins, and in some cases, cause severe, incurable diseases of humans and animals.
The previously unknown widespread occurrence of prion-like proteins and proteins with prion-like domains among animals, humans, bacteria, archaea, fungi and viruses makes their detection relevant for diagnostic purposes, and moreover might be an important approach for the therapy and prevention of various diseases.
Recently, prions and their infectious forms have attracted a lot of research attention (Eisenberg and Jucker, 2012; Morales, 2017). The infectious prion forms (PrPSc) represent the misfolded normal proteins (PrPC) and were shown to be infectious, since they can self-propagate and interact with the endogenous PrPC, catalyzing their conversion into pathological PrPScs (Prusiner 1998; Ma, 2002; Stefani, 2004; Cobb and Surewicz, 2009). PrPSc had been primarily known as inducers of transmissible spongiform encephalopathies, however, today they have been shown to be involved in the development of a variety of neurodegenerative diseases (Goedert et al., 2010; Furukawa and Nukina, 2013; Prusiner, 2013).
Prion proteins (PrPs) are characterized by self-propagation, undergoing a conformational switch from one conformational state to another which leads to the creation of new prions. Pathologically, prions are characterized by a process in which the infectious form of prion (PrPSc) interacts with the endogenous PrPs, catalyzing the transformation of the endogenous molecule into misfolded PrPSc aggregates.
Many PrPs contain prioniogenic domains (PrDs), whose functionalities and distribution in different viral families and species have not be determined to date.
In one aspect is provided a method of diagnosing a disease in a subject, which method comprises: a) heating a sample collected from the subject for 10 seconds to 48 hours at a temperature from 43° C. to 200° C., b) isolating a soluble protein fraction in the sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) comparing the level of the one or more polypeptides identified in step (c) with a control level(s) of said polypeptide(s), and e) (i) identifying the subject as being afflicted with the disease when the level(s) of said one or more polypeptides is different by 10% or more from the control level(s), or (ii) identifying that the subject is not afflicted with the disease if the level(s) of said one or more polypeptides differs from the control level(s) by less than 10%.
In another aspect is provided a method of monitoring changes in development of a disease in a subject, which method comprises: a) heating a first sample collected from the subject for 10 seconds to 48 hours at a temperature from 43° C. to 200° C., b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject at later time points than the first sample, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the disease has progressed when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that the disease has not progressed when the level(s) of the one or more polypeptides identified in step (d) is not higher than the level(s) of said polypeptide(s) identified in step (c).
In another aspect is provided a method of monitoring the effect of a treatment on development of a disease in a subject who had been previously diagnosed with the disease, which method comprises: a) heating a first sample collected from the subject for 10 seconds to 48 hours at a temperature from 43° C. to 200° C., wherein said first sample has been collected from the subject prior to initiation of the treatment, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject after initiation of the treatment, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the treatment is effective when the level(s) of the one or more polypeptides identified in step (d) is the same or lower than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that treatment is not effective when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c).
In another aspect is provided a method for identifying a compound useful for slowing down the progression or treating a disease in a subject who had been previously diagnosed with the disease, which method comprises: a) heating a first sample collected from the subject for 10 seconds to 48 hours at a temperature from 43° C. to 200° C., wherein said first sample has been collected from the subject prior to administration of a test compound, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject after administration of the test compound, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the test compound is useful for slowing down the progression or treating the disease when the level(s) of the one or more polypeptides identified in step (d) is the same or lower than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that the test compound is not useful for slowing down the progression or treating the disease when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c).
In some embodiments of any of the above methods, the sample is heated for 5 seconds to 15 minutes at 80-120° C. In one embodiment, the sample is heated for 5 seconds to 15 minutes at about 100° C. In one embodiment, the sample is heated for about 30 minutes at about 50° C. In one embodiment, the method further comprises adding a polynucleotide molecule to the sample. In one embodiment, the polynucleotide molecule is added to the sample after step (a) and before step (b). In one embodiment, the polynucleotide molecule is added to the sample before step (a). In one embodiment, the polynucleotide molecule is DNA. In one embodiment, the polynucleotide molecule is RNA. In one embodiment, the sample is incubated in the presence of the polynucleotide molecule for 1 minute to 72 hours at 20-60° C. In one embodiment, the sample is incubated in the presence of the polynucleotide molecule for 30 minutes to 5 hours at 30-40° C. In one embodiment, the sample is incubated in the presence of the polynucleotide molecule for about 10 to 120 minutes at about 37° C.
In one embodiment, the polynucleotide molecule is added at the final concentration of 0.1 ng/ml to 2000 μg/ml. In one embodiment, the polynucleotide molecule is added at the final concentration of 100-10000 ng/ml. In one embodiment, the method further comprises adding a protease.
In one embodiment, the protease is added after step (a) and before step (b). In one embodiment, the protease is added before step (a). In one embodiment, the sample is incubated in the presence of the protease for 30 seconds to 5 days at 20-200° C. In one embodiment, the sample is incubated in the presence of the protease for about 30 minutes to 5 hours at 30-40° C. In one embodiment, the sample is incubated in the presence of the protease for about 10 to 120 minutes at about 37° C.
In various embodiments of the above methods, the protease is proteinase K.
In various embodiments of the above methods, the protein fraction is a soluble protein fraction. In various embodiments of the above methods, the protein fraction is an insoluble protein fraction.
In another aspect is provided a method of diagnosing a disease in a subject, which method comprises: a) adding to a sample collected from the subject a polynucleotide molecule and incubating the sample with said polynucleotide molecule, b) isolating a protein fraction in the sample, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) comparing the level of the one or more polypeptides identified in step (c) with a control level(s) of said polypeptide(s), and e) (i) identifying the subject as being afflicted with the disease when the level of said one or more polypeptides is different by 10% or more from the control level, or (ii) identifying that the subject is not afflicted with the disease if the level of said one or more polypeptides differs from the control level by less than 10%.
In another aspect is provided a method of monitoring changes in development of a disease in a subject, which method comprises: a) adding to a first sample collected from the subject a polynucleotide molecule and incubating the sample with said polynucleotide molecule, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject at later time points than the first sample, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the disease has progressed when the level(s) of the one or more polypeptidesidentified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that the disease has not progressed when the level(s) of the one or more polypeptides identified in step (d) is not higher than the level(s) of said polypeptide(s) identified in step (c).
In another aspect is provided a method of monitoring the effect of a treatment on development of a disease in a subject who had been previously diagnosed with the disease, which method comprises: a) adding to a first sample collected from the subject a polynucleotide molecule and incubating the sample with said polynucleotide molecule, wherein said first sample has been collected from the subject prior to initiation of the treatment, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject after initiation of the treatment, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the treatment is effective when the level(s) of the one or more polypeptides identified in step (d) is the same or lower than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that treatment is not effective when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c).
In another aspect is provided a method for identifying a compound useful for slowing down the progression or treating a disease in a subject who had been previously diagnosed with the disease, which method comprises: a) adding to a first sample collected from the subject a polynucleotide molecule and incubating the sample with said polynucleotide molecule, wherein said first sample has been collected from the subject prior to administration of a test compound, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject after administration of the test compound, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the test compound is useful for slowing down the progression or treating the disease when the level(s) of the one or more polypeptides identified in step (d) is the same or lower than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that the test compound is not useful for slowing down the progression or treating the disease when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c).
In some embodiments, the polynucleotide molecule is DNA. In some embodiments, the polynucleotide molecule is RNA. In some embodiments, the sample is incubated in the presence of the polynucleotide molecule for 1 minute to 72 hours at 20-60° C. In some embodiments, the sample is incubated in the presence of the polynucleotide molecule for 30 minutes to 5 hours at 30-40° C. In some embodiments, the sample is incubated in the presence of the polynucleotide molecule for about 1 minute to 24 hours at about 37° C. In some embodiments, the polynucleotide molecule is added at the final concentration of 0.1 ng/ml to 2000 μg/ml. In some embodiments, the polynucleotide molecule is added at the final concentration of 100-10000 ng/ml.
In another aspect is provided a method of diagnosing a disease in a subject, which method comprises: a) adding to a sample collected from the subject a protease and incubating the sample with said protease, b) isolating a soluble protein fraction in the sample, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) comparing the level of the one or more polypeptides identified in step (c) with a control level(s) of said polypeptide(s), and e) (i) identifying the subject as being afflicted with the disease when the level of said one or more polypeptides is different by 10% or more from the control level, or (ii) identifying that the subject is not afflicted with the disease if the level of said one or more polypeptides differs from the control level by less than 10%.
In another aspect is provided a method of monitoring changes in development of a disease in a subject, which method comprises: a) adding to a first sample collected from the subject a protease and incubating the sample with said protease, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject at later time points than the first sample, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the disease has progressed when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that the disease has not progressed when the level(s) of the one or more polypeptides identified in step (d) is not higher than the level(s) of said polypeptide(s) identified in step (c).
In another aspect is provided a method of monitoring the effect of a treatment on development of a disease in a subject who had been previously diagnosed with the disease, which method comprises: a) adding to a first sample collected from the subject a protease and incubating the sample with said protease, wherein said first sample has been collected from the subject prior to initiation of the treatment, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject after initiation of the treatment, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the treatment is effective when the level(s) of the one or more polypeptides identified in step (d) is the same or lower than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that treatment is not effective when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c).
In another aspect is provided a method for identifying a compound useful for slowing down the progression or treating a disease in a subject who had been previously diagnosed with the disease, which method comprises: a) adding to a first sample collected from the subject a protease and incubating the sample with said protease, wherein said first sample has been collected from the subject prior to administration of a test compound, b) isolating a soluble protein fraction in the first sample after the completion of the heating, c) determining the level of one or more polypeptides in the soluble protein fraction isolated in step (b), d) repeating steps (a)-(c) for one or more additional samples, wherein said additional samples have been collected from the subject after administration of the test compound, e) comparing the levels of the one or more polypeptides identified in step (c) with the level(s) of said polypeptide(s) identified in step (d), and f) (i) determining that the test compound is useful for slowing down the progression or treating the disease when the level(s) of the one or more polypeptides identified in step (d) is the same or lower than the level(s) of said polypeptide(s) identified in step (c), or (ii) determining that the test compound is not useful for slowing down the progression or treating the disease when the level(s) of the one or more polypeptides identified in step (d) is higher than the level(s) of said polypeptide(s) identified in step (c).
In some embodiments, the sample is incubated in the presence of the protease for 30 seconds to 5 days at 20-200° C. In some embodiments, the sample is incubated in the presence of the protease for about 30 minutes to 5 hours at 30-40° C. In some embodiments, the sample is incubated in the presence of the protease for about 1 minute to 24 hours at about 37° C. In some embodiments, the protease is proteinase K. In some embodiments, the polypeptide is a full-length protein or a full-length subunit of a protein complex. In some embodiments, the polypeptide is a fragment of a full-length protein.
In some embodiments, the fragment of a full-length protein is a domain of said full-length protein.
In various embodiments of the above methods, the control level is a predetermined value. In some embodiments, the control level is the level of said polypeptide in a similarly processed bodily fluid sample of one or more age-matched healthy subjects. In some embodiments, control level is the level of said polypeptide in a similarly processed bodily fluid sample from the same subject collected in the past.
In various embodiments of the above methods, the soluble protein fraction is isolated by one or more of the methods selected from centrifugation, filtering, treatment with a detergent, rehydration, protein extraction, and treatment with a chaotropic buffer. In some embodiments, the detergent is SDS.
In various embodiments of the above methods, the polynucleotide comprises from 2 to 1,000,000 nucleotides or base pairs. In various embodiments of the above methods, the polynucleotide is from 10 base pairs to 1,000,000 nucleotides or base pairs. In various embodiments of the above methods, the polynucleotide molecule is of human, viral or bacterial origin.
In various embodiments of the above methods, the one or more of the polypeptides are selected from the proteins listed in Table 4, 5, 6, 7, 8, 10, 11, 19, 23, 24, or 27.
In various embodiments of the above methods, the levels of two or more polypeptides are measured. In some embodiments, the levels of five or more polypeptides are measured. In some embodiments, the levels of twenty or more polypeptides are measured.
In various embodiments of the above methods, the polypeptide level is calculated as the sum of each of the measured polypeptide levels. In some embodiments, the sum of each of the measured polypeptide levels is weighted.
In various embodiments of the above methods, the sample is selected from a bodily fluid sample, cells, cell lysate, tissue sample, tumor sample, and a microbial biofilm matrix. In some embodiments, the bodily fluid sample is selected from whole blood, plasma, serum, cerebrospinal fluid, amniotic fluid, urine, and saliva.
In various embodiments of the above methods, the subject is human.
In various embodiments of the above methods, the disease is selected from a cancer, an infection, a neurodegenerative disease, a neurodevelopmental disease, an abnormal pregnancy, aging, and an autoimmune disease.
In various embodiments of the above methods, the one or more of the polypeptides does not comprise prion-like domains.
In various embodiments of the above methods, the one or more of the polypeptides is a Tetz-protein or a fragment thereof. In some embodiments, the Tetz-protein is a thermostable Tetz-protein. In some embodiments, the Tetz-protein is a non-thermostable Tetz-protein. In some embodiments, the Tetz-protein is a bacterial, archaeal, fungal, or viral protein. In some embodiments, the virus is a bacteriophage or an animal virus.
In various embodiments of the above methods, the one or more of the polypeptides is a prion-like protein or a fragment thereof. In some embodiments, the prion-like protein is a bacterial, archaeal, fungal, or viral protein. In some embodiments, the virus is a bacteriophage or an animal virus.
In various embodiments of the above methods, the one or more of the polypeptides comprises a prion-like domain (PrD). In some embodiments, the polypeptide is a bacterial, archaeal, fungal, or viral protein. In some embodiments, the virus is a bacteriophage or an animal virus.
In various embodiments of the above methods, the level of one or more polypeptides is determined using one or more methods selected from electrophoresis, chromatography, an immunoassay, mass spectrometry, and methods involving dyes.
In various embodiments of the above methods, the disease is cancer and the method comprises measuring the level of one or more proteins listed in Table 4, 5, 6, 7, 8, 9, 10, 11, 19, 23, or 24. In some embodiments, the method comprises determining the level of one or more proteins listed in Tables 5, 7, 8, or 9.
In various embodiments of the above methods, the disease is cancer and the method comprises measuring the level of one or more proteins selected from serum albumin, Fibronectin, Complement factor B, Vitamin D-binding protein, Immunoglobulin heavy constant gamma 2, Plasminogen, Inter-alpha-trypsin inhibitor heavy chain H4, Inter-alpha-trypsin inhibitor heavy chain H2, Apolipoprotein B-100, Apolipoprotein L1, Alpha-1-acid glycoprotein 2, C4b-binding protein beta chain, Immunoglobulin heavy constant gamma 1, Apolipoprotein A-II, Alpha-1-acid glycoprotein 2, Apolipoprotein B-100, Hemoglobin subunit alpha, CD5 antigen-like, Selenoprotein P, Immunoglobulin lambda constant 3, Eukaryotic translation initiation factor 5A-1, Cluster of Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 9, Keratin, type I cytoskeletal 10, Immunoglobulin kappa variable 1-27, Chromodomain-helicase-DNA-binding protein 7, Fetuin-B, Immunoglobulin heavy constant gamma 1, Immunoglobulin heavy constant gamma 4, Immunoglobulin lambda variable 3-27, Kallikrein-2, N-lysine methyltransferase SETD6, Protein SON, Reversion-inducing cysteine-rich protein with Kazal motifs, CON_Q2UVX4, Serotransferrin, Gelsolin, Complement C2, Complement factor H-related protein 1, Pigment epithelium-derived factor, Hemoglobin subunit alpha, Complement C5, Complement C1q, Immunoglobulin lambda constant 7, Actin, cytoplasmic 1, Coagulation factor XII, Complement component C6, Calmodulin-1, Tropomyosin alpha-4, Tropomyosin beta Epididymis luminal protein 189, Tropomyosin alpha-1, and Tropomyosin alpha-3.
In various embodiments of the above methods, the disease is cancer and the method comprises measuring the level of one or more proteins selected from CON_Q2UVX4, Serotransferrin, Complement factor H-related protein 1, Pigment epithelium-derived factor, Cluster of Hemoglobin subunit alpha, Hemoglobin subunit alpha, CON_P01966, Complement C5, and Immunoglobulin lambda constant 7.
In various embodiments of the above methods, the disease is cancer and the method comprises measuring the level of one or more proteins selected from Complement C3, CON_Q2UVX4, Serotransferrin, Gelsolin, Immunoglobulin lambda constant 7, and Inter-alpha-trypsin inhibitor heavy chain H3.
In various embodiments of the above methods, the disease is a neurodegenerative, neurodevelopmental or congenital disease.
In various embodiments of the above methods, the method further comprises administering a treatment to the subject. In some embodiments, the treatment involves inhibiting expression or activity of the said one or more polypeptides. In some embodiments, the treatment involves exposure to polypeptide-specific antibodies and/or highly-specific protease treatment. In some embodiments, the one or more polypeptides comprises a prion-like domain (PrD) and the antibodies interact with said PrD. In some embodiments, the treatment involves destruction of extracellular DNA. In some embodiments, the destruction of extracellular DNA involves treatment with a DNase. In some embodiments, the treatment involves administering said one or more polypeptides to the subject.
In some embodiments, the one or more polypeptides comprises a prion-like domain (PrD) and the treatment comprises administering an effective amount of an anti-PrD drug to the subject. In one embodiment, the disease is an infection selected from a viral infection, a bacterial infection, a fungal infection, and a protozoal infection. In one embodiment, the disease is a neurodegenerative disorder. In one embodiment, the disease is selected from scrapie, Creutzfeldt-Jakob disease, Alzheimer's disease, Parkinson's disease, amyloidosis, Huntington's disease, fatal familial insomnia, ataxias, a dementia, amyotrophic lateral sclerosis, CADASIL, and diabetes.
In some embodiments, the anti-PrD drug is selected from tacrolimus, pentosan polysulfate, quinacrine, an antibody against an amyloid protein, an antibody against a nuclease, an antibody against a protease, and rituximab. In one embodiment, the antibody against an amyloid protein is an antibody against beta amyloid. In one embodiment, the antibody against a nuclease is an antibody against a DNase. In one embodiment, the antibody against a protease is an antibody against proteinase K.
In another aspect is provided a method of diagnosing a viral infection in a subject, which method comprises: a) treating a sample collected from the subject with an antibody against a prion-like domain (PrD) or a protein comprising a PrD, wherein said PrD or protein is present in said virus, and b) identifying the subject as being afflicted with the viral infection when an increased reactivity of the antibody to the PrD or the protein comprising the PrD is detected in the sample collected from the subject as compared to the antibody reactivity in a control. In some embodiments, the protein comprising the PrD is selected from the proteins recited in Table 15.
In another aspect is provided a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of a compound which inhibits expression or activity of one or more polypeptides selected from thermostable Tetz-proteins, non-thermostable disease-associated Tetz-proteins, proteins comprising PrDs, prion-like proteins, and fragments thereof. In some embodiments, the thermostable Tetz-proteins are heat-resistant Tetz-proteins. In some embodiments, the disease is selected from a cancer, an infection, a neurodegenerative disease, a neurodevelopmental disease, an abnormal pregnancy, aging, and an autoimmune disease. In some embodiments, the infection is selected from a viral infection, a bacterial infection, a fungal infection, and a protozoal infection. In some embodiments, the disease is selected from scrapie, Creutzfeldt-Jakob disease, Alzheimer's disease, Parkinson's disease, amyloidosis, Huntington's disease, fatal familial insomnia, ataxias, and diabetes.
In another aspect is provided a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound which inhibits expression or activity of one or more proteins listed in Table 4, 5, 6, 7, 8, 9, 10, 11, 19, 23, or 24. In some embodiments, the compound inhibits expression or activity of one or more proteins listed in Tables 5, 7, 8, or 9.
In another aspect is provided a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound which inhibits expression or activity of one or more proteins selected from serum albumin, Fibronectin, Complement factor B, Vitamin D-binding protein, Immunoglobulin heavy constant gamma 2, Plasminogen, Inter-alpha-trypsin inhibitor heavy chain H4, Inter-alpha-trypsin inhibitor heavy chain H2, Apolipoprotein B-100, Apolipoprotein L1, Alpha-1-acid glycoprotein 2, C4b-binding protein beta chain, Immunoglobulin heavy constant gamma 1, Apolipoprotein A-II, Alpha-1-acid glycoprotein 2, Apolipoprotein B-100, Hemoglobin subunit alpha, CD5 antigen-like, Selenoprotein P, Immunoglobulin lambda constant 3, Eukaryotic translation initiation factor 5A-1, Cluster of Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 9, Keratin, type I cytoskeletal 10, Immunoglobulin kappa variable 1-27, Chromodomain-helicase-DNA-binding protein 7, Fetuin-B, Immunoglobulin heavy constant gamma 1, Immunoglobulin heavy constant gamma 4, Immunoglobulin lambda variable 3-27, Kallikrein-2, N-lysine methyltransferase SETD6, Protein SON, Reversion-inducing cysteine-rich protein with Kazal motifs, CON_Q2UVX4, Serotransferrin, Gelsolin, Complement C2, Complement factor H-related protein 1, Pigment epithelium-derived factor, Hemoglobin subunit alpha, Complement C5, Complement C1q, Immunoglobulin lambda constant 7, Actin, cytoplasmic 1, Coagulation factor XII, Complement component C6, Calmodulin-1, Tropomyosin alpha-4, Tropomyosin beta Epididymis luminal protein 189, Tropomyosin alpha-1, and Tropomyosin alpha-3.
In another aspect is provided a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound which inhibits expression or activity of one or more proteins selected from serum albumin, Fibronectin, Complement factor B, Vitamin D-binding protein, Immunoglobulin heavy constant gamma 2, Plasminogen, Inter-alpha-trypsin inhibitor heavy chain H4, Inter-alpha-trypsin inhibitor heavy chain H2, Apolipoprotein B-100, Apolipoprotein L1, Alpha-1-acid glycoprotein 2, C4b-binding protein beta chain, Immunoglobulin heavy constant gamma 1, Apolipoprotein A-II, Alpha-1-acid glycoprotein 2, Apolipoprotein B-100, Hemoglobin subunit alpha, CD5 antigen-like, Selenoprotein P, Immunoglobulin lambda constant 3, Eukaryotic translation initiation factor 5A-1, Cluster of Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 9, Keratin, type I cytoskeletal 10, Immunoglobulin kappa variable 1-27, Chromodomain-helicase-DNA-binding protein 7, Fetuin-B, Immunoglobulin heavy constant gamma 1, Immunoglobulin heavy constant gamma 4, Immunoglobulin lambda variable 3-27, Kallikrein-2, N-lysine methyltransferase SETD6, Protein SON, Reversion-inducing cysteine-rich protein with Kazal motifs, CON_Q2UVX4, Serotransferrin, Complement factor H-related protein 1, Pigment epithelium-derived factor, Cluster of Hemoglobin subunit alpha, Hemoglobin subunit alpha, CON_P01966, Complement C5, and Immunoglobulin lambda constant 7.
In another aspect is provided a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound which inhibits expression or activity of one or more proteins selected from serum albumin, Fibronectin, Complement factor B, Vitamin D-binding protein, Immunoglobulin heavy constant gamma 2, Plasminogen, Inter-alpha-trypsin inhibitor heavy chain H4, Inter-alpha-trypsin inhibitor heavy chain H2, Apolipoprotein B-100, Apolipoprotein L1, Alpha-1-acid glycoprotein 2, C4b-binding protein beta chain, Immunoglobulin heavy constant gamma 1, Apolipoprotein A-II, Alpha-1-acid glycoprotein 2, Apolipoprotein B-100, Hemoglobin subunit alpha, CD5 antigen-like, Selenoprotein P, Immunoglobulin lambda constant 3, Eukaryotic translation initiation factor 5A-1, Cluster of Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 9, Keratin, type I cytoskeletal 10, Immunoglobulin kappa variable 1-27, Chromodomain-helicase-DNA-binding protein 7, Fetuin-B, Immunoglobulin heavy constant gamma 1, Immunoglobulin heavy constant gamma 4, Immunoglobulin lambda variable 3-27, Kallikrein-2, N-lysine methyltransferase SETD6, Protein SON, Reversion-inducing cysteine-rich protein with Kazal motifs, Complement C3, CON_Q2UVX4, Serotransferrin, Gelsolin, Immunoglobulin lambda constant 7, and Inter-alpha-trypsin inhibitor heavy chain H3.
In some embodiments of the above aspects on methods of treating, the treatment involves exposure to polypeptide-specific antibodies and/or highly-specific protease treatment. In some embodiments, the one or more polypeptides comprises a prion-like domain (PrD) and the antibodies interact with said PrD. In some embodiments, the treatment involves destruction of extracellular DNA. In some embodiments, the destruction of extracellular DNA involves treatment with a DNase. In some embodiments, the treatment involves administering to the subject an effective amount of an anti-PrD drug. In some embodiments, the anti-PrD drug is selected from tacrolimus, pentosan polysulfate, quinacrine, an antibody against an amyloid protein, an antibody against a nuclease, an antibody against a protease, and rituximab. In some embodiments, the antibody against an amyloid protein is an antibody against beta amyloid. In some embodiments, the antibody against a nuclease is an antibody against a DNase. In some embodiments, the antibody against a protease is an antibody against proteinase K.
In another aspect is provided a method of treating a disease in a human subject comprising administering to the subject an effective amount of a compound that inhibits a human cell or a human protein from interacting with a viral protein comprising a prion-like domain. In another aspect is provided method of treating a disease in a human subject comprising administering to the subject an effective amount of a compound that prevents a human cell or a human protein from interating with a viral protein comprising a prion-like domain.
In some embodiments of the above aspects on methods of treating a disease in a human subject, the disease is a cancer or a neurodegenerative disease. In some embodiments, the viral protein comprising a prion-like domain is a viral protein from HIV, HHV-1, HHV-5, HHV-6, or HIV-8. In some embodiments, the viral protein comprising a prion-like domain is HIV-1 envelope glycoprotein gp160 (E5RVW7), Gag protein (C1JH95), Pol protein (Q3S7Q7), Envelope glycoprotein gp120 (Q2ME99), Human herpes simplex virus 8 RF1 (U5NM22); Human herpes simplex virus 8 LANA (E5LC01), Human herpes simplex virus 8 ORF 73 (A0A0N9S3L8), Human herpes simplex virus 6 (U95 protein), Human herpes simplex virus 1 large tegument protein deneddylase, Human herpes simplex virus 1 envelope glycoprotein I, Human herpes simplex virus 1 envelope glycoprotein 2, or Varicella zoster small capsomere-interacting protein. In some embodiments, the viral protein comprising the prion-like domain is capable of altering a prionogenic-like protein. In some embodiments, the viral protein comprising the prion-like domain is capable of misfolding the prionogenic-like protein. In some embodiments, the prionogenic-like protein is Tau proterin, betta-amyloid, P53, SOD1, TDP43, or alpha-synuclein.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The terms “prion-like domain (PrD)” or “prionogenic domain” are used herein to refer to parts of a protein that can become a Prion protein (PrP). PrPs are characterized by self-propagation, and undergo a conformational switch from one conformational state to another, which leads to the creation of new prions.
As used herein, the term “Tetz-proteins” encompasses: (i) thermostable proteins, (ii) fragments or domains of thermostable proteins, (iii) thermostable fragments or domains of non-thermostable proteins, and (iv) thermostable protein subunits of non-thermostable protein complexes, wherein said proteins, protein subunits, fragments and domains are not prions, do not comprise prion-like domains, and remain in a soluble protein fraction after heating a sample containing such proteins, protein subunits, fragments and domains (e.g., a bodily fluid sample collected from a subject) for, e.g., about 10 minutes to 8 hours at about 50° C., or about 30 seconds to 8 hours at about 100° C., or 5 minutes to 8 hours at 80-120° C. In addition to the above thermostable proteins, protein subunits, fragments and domains, the term “Tetz-proteins” also encompasses (v) non-thermostable proteins, (vi) fragments or domains of non-thermostable proteins, and (vii) proteins having a structure (e.g., tertiary or quaternary structure) found in mesophilic or psychrophilic organisms, wherein said proteins, fragments or domains (v)-(vii) are associated with a pathology, are not prions, do not comprise prion-like domains, and are formed or their amount is increased in the presence of nucleic acids (e.g., DNA or RNA, e.g., ranging in size from 10 bp to 1,000,000 bp) and/or a protease (e.g., proteinase K). Thermostable Tetz-proteins, protein subunits, fragments and domains (i)-(iv) also can be (but do not have to be) formed or their amount can be increased in the presence of nucleic acids (e.g., DNA or RNA, e.g., ranging in size from 10 bp to 1,000,000 bp) and/or a protease (e.g., proteinase K).
Tetz-proteins can be found in and identified in bodily fluids (e.g., whole blood, plasma, serum, cerebrospinal fluid, amniotic fluid, urine, or saliva), cells, cell lysates, and microbial biofilm matrices.
“Targets” are molecules with which prion-like and/or Tetz-proteins can interact and/or bind. Protein targeted DNA (ptDNA) includes DNA forming a complex with extracellular proteins and DNA that changes the properties of extracellular proteins. Protein targeted RNA (ptRNA) includes RNA forming a complex with extracellular proteins and RNA that changes the properties of extracellular proteins.
As used herein, the term “therapeutically effective amount” refers to the amount of a compound, composition, particle, organism (e.g., a probiotic or a microbiota transplant), etc. that, when administered to a subject for treating (e.g., preventing or ameliorating) a state, disorder or condition, is sufficient to effect such treatment. The “therapeutically effective amount” will vary depending, e.g., on the agent being administered as well as the disease severity, age, weight, and physical conditions and responsiveness of the subject to be treated.
As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally regarded as physiologically tolerable.
The terms “patient”, “individual”, “subject”, “mammal”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.
The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
The term “about” or “approximately” means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
The terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J. Additional techniques are explained, e.g., in U.S. Pat. No. 7,912,698 and U.S. Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437. In some embodiments, proteins comprising PrDs comprise glutamine/asparagine (Q/N) enriched PrDs. In some embodiments, PrDs are determined using protein analysis (e.g., Western blot, ELISA) and/or algorithms (e.g., PLAAC algorithm, a web and command-line application to identify proteins with Prion-Like Amino Acid Composition Bioinformatics, and an algorithm using an experimentally-derived prion propensity score combined with explicit consideration of the intrinsic disorder so as to bioinformatically predict prion domains, such as PAPA and PrionW).
The studies described herein are the most complete evaluation of PrDs among viruses except for the bacteriophages. The results highlight some previously overlooked viral characteristics that may play important roles in viral infections.
PrDs were identified in functionally distinct proteins of different viral orders, indicating that these PrDs are conserved in different viruses. However, the PrDs were not identified in all viral families and species. The above analyses demonstrate that only approximately 23% of all analyzed viral proteomes available in public databases contain at least one PrD. PrDs were identified in many human viral pathogens, but other viruses affecting human health were shown to have a few or no PrDs in their proteomes, such as hepatitis A, E, and D viruses, papillomaviruses, some members of Orthomyxoviridae, and others.
At the order level, PrDs are more frequent among Megavirales and Herpesvirales, while at the species level, the highest number of PrDs was found in Acanthamoeba polyphaga mimivirus, Paramecium bursaria Chlorella virus NY2A, Acanthamoeba castellanii mamavirus (Megavirales), and Heliothis zea nudivirus (unassigned order). Among human pathogens, the highest prevalence of PrD was found in cytomegalovirus and Epstein-Barr virus (Herpesvirales) and HIV1 (Retroviridae family, unassigned order).
In an analysis of the top 100 scoring PrDs with the highest number of QN-rich domains, such top scoring PrDs were found to be most common among Mimiviridae, which infect Acanthamoeba, and Phycodnaviridae, which infect algae and belong to the Megavirales. Of these, only some proteins were Herpesvirales proteins, while the majority of them was shown to be identified in the viruses of the unassigned order. No human viruses were shown to have log-likelihood ratio (LLR) scores over 31 and none were represented in the top 100 LLR-scoring group. (The LLR score reflects the similarity between the examined interaction sets, with an LLR near zero suggesting a comparison of sets of random interactions.) The majority of these proteins has not been characterized.
The order Megavirales is a recently established order that comprises of diverse group of the DNA-viruses infecting eukaryotic hosts, which are characterized by large genomes. Here, DNA-viruses were found to harbor more high-scoring prions, as expected, but the high LLR scores obtained for these viruses is not due to the longer amino-acid sequences, but to the increased presence of QN-residues.
Furthermore, the inventors aimed to determine the correlation between the PrD-containing protein functions and the frequency of PrDs in the viral proteomes found in different viral families. Adhesion and entry of viral nucleic acids represent crucial steps in the viral-host interactions and the viral PrD-containing proteins showed to be involved in these processes represented the second largest group. PrDs in the viral surface proteins were identified that are involved in the direct contact and fusion of viruses with the host cell membrane, indicating that PrDs may be functionally implicated in these processes as well.
Of 543 PrDs found to be associated with the viral interaction with the host cells, only four proteins were identified in the plant viruses (potato mop-top virus, Dasheen mosaic virus, only Syngen Nebraska virus 5, and Fiji disease virus). Plant viruses are known to have no specific mechanisms of entry, but instead they take advantage of the plant injury, vectors such as insects, or through a cell-to-cell movement of viral progeny in the infected plant (Wolf et al., 1989; Dasgupta et al., 2017; Ackermann, 2017). PrDs present in the proteins of animal viruses that interact with cell membranes may be associated with adhesion and entry, and may have important functional roles.
Taken together, numerous putative PrD-containing proteins were identified in viruses. Consistent PrD distribution patterns were observed in different viral families and species, and these domains were identified in a variety of proteins. Without wishing to be bound by theory, the majority of viruses were shown to lack the PrDs, which shows that the presence of PrDs is beneficial, but not obligatory, and which agrees with the results obtained for the PrDs found in bacteria and bacteriophages. The predictive approach employed in this study revealed for the first time a large set of putative PrPs in numerous proteins of the emerging human viral pathogens, including those associated with persistent viral infections, oncogenic processes, hemorrhagic fevers, and others. Further analyses of these PrD-containing proteins may contribute to the better understanding of viral infections. Also, gene editing may be used as a technology that could allow for development of viruses with an advanced prion-like domain profile on their surfaces.
Without wishing to be bound by theory, PrDs in viral proteins may be important for assembly and growth of viral capsids. PrDs may be involved in liquid-liquid phase separation (LLPS), and in turn the nucleation and growth of protein crystals. Further, LLPS may play a role in the first steps of viral capsid growth. PrD-containing proteins may promote or enhance LLPS, and thus promote assembly of the viral capsid by scaffolding proteins.
Tetz-proteins may be identified by obtaining a bodily fluid sample from a patient, such as blood plasma. The blood plasma may be untreated, treated with a nucleotide (e.g., 0.01-10000 mcg/ml DNA), treated with a protease (e.g., 0.01-10000 mcg/ml proteinase K), or treated with both the nucleotide and the protease. The blood plasma may be heated at a temperature from 43 to 200° C. for 20 seconds minute to 5 hours.
Subsequently, the bodily fluid sample may be subjected to abundant protein depletion so as to remove abundant proteins from serum or plasma samples. A kit may be used, such as the ProteoSpin™ Abundant Serum Protein Depletion Kit available from Norgen Biotek and the Seppro® Protein Depletion kit available from Sigma-Aldrich.
Proteins may then be separated by gel electrophoresis or liquid chromatography, and then analyzed by mass spectrometry. Isobaric labeling at the peptide level for multiplexed relative quantification may be undertaken. SELDI-TOF mass spectrometry may be used. Also, ultra-high performance liquid chromatography may be coupled to accurate-mass high resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography.
In one embodiment of the various aspects and embodiments described in the application, the disease is a neurodegenerative disorder.
In one embodiment of the various aspects and embodiments described in the application, the disease is scrapie, Creutzfeldt-Jakob disease, Alzheimer's disease, Parkinson's disease, amyloidosis, bipolar disorder, depressive disorder, schizophrenia, Huntington's disease, fatal familial insomnia, Chronic Fatigue Syndrome, a dementia, generalized anxiety disorder (GAD), major depressive disorder (MDD), multiple sclerosis, CADASIL Syndrome, an ataxia, a Lewy body disease, social anxiety disorder (SAD), attention-deficit/hyperactivity disorder (ADHD), autism and autism spectrum disorder, amyotrophic lateral sclerosis, α-Synucleinopathies and diabetes, a renal disorder (e.g., primary membranoproliferative glomerulonephritis, immunoglobulin-mediated membranoproliferative glomerulonephritis, non-immunoglobulin-mediated membranoproliferative glomerulonephritis, fibronectin glomerulopathy, primary glomerular disease, dense deposit disease), one or more eye disorders; one or more hematologic diseases; an intestinal disorder, a heart disorder, one or more nervous system disorders; hyperthyroxinemia, glioma, schizophrenia, Ehlers-Danlos syndrome, otopalatodigital syndrome, Noonan syndrome, Erythroderma desquamativum, cancer, aging, an age-related change of the skin, rheumatoid arthritis, atopic dermatitis, ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), scleroderma, liver failure, liver cirrhosis, chronic heart failure, atherosclerosis, myocardial infarction, thrombosis, gout, one or more cancers, cancer cachexia, graft-versus-host reactions, rhythm and conduction disturbances, primary biliary cirrhosis, primary sclerosing cholangitis, and asthma.
In one embodiment, the renal disorder is atypical hemolytic-uremic syndrome.
In one embodiment, the eye disorder is retinal dystrophy, age-related macular degeneration, corneal dystrophy, familial drusen, or ligneous conjunctivitis.
In one embodiment, the hematologic disease is congenital atransferrinemia, hypochromic anemia, α-thalassemia, Hb Bart's hydrops fetalis, lymphedema, an immunodeficiency due to a complement cascade protein anomaly, a hypoplasminogenemia, AL amyloidosis, familial amyloidosis Finnish type, or a gamma 1 chain deposition disease.
In one embodiment, the intestinal disorder is congenital sodium diarrhea, chronic intestinal pseudoobstruction, or congenital short bowel syndrome.
In one embodiment, the heart disorder is dilated cardiomyopathy, coronary artery disease, or hypertrophic cardiomyopathy.
In one embodiment, the cancer is lung cancer, ovarian cancer, astrocytoma, non-cell small lung cancer, pancreatic cancer, thyroid carcinoma, or lung carcinoma.
In one embodiment, the nervous system disorder is neurodegeneration with brain iron accumulation, nodular neuronal heterotopia, or aceruloplasminemia.
In one embodiment, the disease is an infection selected from a viral infection, a bacterial infection, a fungal infection, and a protozoal infection.
1. A method for producing vectors, vaccines, diagnosis, treatment and prevention of diseases, characterized in that the prion-like and Tetz-proteins or the molecules involved in their formation and/or alteration of their properties and/or interaction with these proteins, are the object of detection and the target of the preventive and treatment preparations.
2. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are in the blood plasma.
3. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are in the blood plasma of humans.
4. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are in the blood plasma of animals.
5. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are in the cells.
6. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are inside human cells.
7. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are inside animal cells.
8. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are in the cerebrospinal fluid.
9. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are in the human cerebrospinal fluid.
10. The method of embodiment 1, wherein the prion-like and/or Tetz-proteins are in the cerebrospinal fluid of animals.
11. Diagnosis of neurodegenerative and neurodevelopmental diseases by identification of viral prion-like and/or Tetz-proteins in the CSF.
12. Diagnosis of neurodegenerative and neurodevelopmental diseases by identification of bacterial prion-like and/or Tetz-proteins in the CSF.
13. Diagnosis of neurodegenerative and neurodevelopmental diseases by identification of human prion-like and/or Tetz-proteins in the CSF.
14. Diagnosis of oncological diseases by identification of for viral prion-like and/or Tetz-proteins in the CSF.
15. Diagnosis of oncological diseases by identification of for bacterial prion-like and/or Tetz-proteins in the CSF.
16. Diagnosis of oncological diseases by identification of for human prion-like and/or Tetz-proteins in the CSF.
17. The method of embodiment 1, wherein prion-like and/or Tetz-proteins are synthesized by bacteria and are within a biofilm matrix.
18. The method of embodiment 1, wherein prion-like and/or Tetz-proteins are synthesized by bacteria and are within the structure of bacterial cells.
19. The method of embodiment 1, wherein prion-like and/or Tetz-proteins are synthesized by archaea and are in biofilm matrix.
20. The method of embodiment 1, wherein prion-like and/or Tetz-proteins are synthesized by archaea and are within archaea cells.
21. The method of embodiment 1, wherein prion-like and/or Tetz-proteins are synthesized by fungi and are in fungal biofilm matrix.
22. The method of embodiment 1, wherein prion-like and/or Tetz-proteins are synthesized by fungi and are within fungal cells.
23. The method of embodiment 1, wherein prion-like and/or Tetz-proteins belong to bacteriophages.
24. The method of embodiment 1, wherein prion-like and/or Tetz-proteins belong to human or animal viruses.
25. The method of embodiment 1, wherein for diagnosis, prion-like and/or Tetz-proteins are detected by protein-detecting methods.
26. The method of embodiment 1, wherein for diagnosis, prion-like and/or Tetz-proteins are detected by electrophoresis.
27. The method of embodiment 1, wherein for diagnosis, prion-like and/or Tetz-proteins are detected by chromatographic methods.
28. The method of embodiment 1, wherein for diagnosis, prion-like and/or Tetz-proteins are detected by Western blot.
29. The method of embodiment 1, wherein for diagnosis, prion-like and/or Tetz-proteins are detected by mass spectrometry.
30. The method of claim 29, wherein the detection by mass spectrometry is by SELDI-TOF mass spectrometry.
31. The method of embodiment 1, wherein for diagnosis, prion-like and/or Tetz-proteins are detected by antibodies.
32. The method of embodiment 1, wherein for diagnosis, prion-like and/or Tetz-proteins are detected by means of dyes.
33. The method of embodiment 1, wherein the composition of thermostable proteins is evaluated for diagnosis of the disease.
34. The method of embodiment 1, wherein for diagnosis of the disease the composition of Tetz-proteins is evaluated, which are detected by heating to temperatures in the range of 50° C. to 250° C.
35. The method of embodiment 1, wherein for diagnosis of the disease the composition of prion-like and/or Tetz-proteins is detected by treatment with proteases.
36. The method of embodiment 1, wherein for diagnosis of the disease the composition of prion-like and/or Tetz-proteins is detected by treatment with a DNA or an RNA, wherein optionally the detection is qualitative or quantitative.
37. The method of embodiment 1, wherein for diagnosis of the disease the composition of prion-like and/or Tetz-proteins, is detected by treatment with bacterial DNA, bacterial RNA, viral DNA, or viral RNA.
38. The method of embodiment 1, wherein for diagnosis of the disease the composition of prion-like and/or Tetz-protein, is detected by treatment with a DNA or an RNA of healthy humans.
39. The method of embodiment 1, wherein for diagnosis of the disease the composition of prion-like and/or Tetz-proteins, which are detected by treatment with a DNA or an RNA of patients with the diagnosed pathology.
40. The method of embodiments 36-39, wherein the disease that is diagnosed is an oncological or neurodegenerative or neurodevelopmental disease.
41. The method of embodiments 36-39, wherein for diagnosis of the disease the is done using the analysis of blood, plasma, serum CSF, amniotic fluid.
42. The method of embodiment 1, wherein prion-like and/or Tetz-proteins found in structures of viruses are detected for the diagnosis of viral infections.
43. The method of embodiment 1, wherein prion-like and/or Tetz-proteins found in structures of bacteria are detected for the diagnosis of viral infections.
44. The method of embodiment 1, wherein prion-like and/or Tetz-proteins found in structures of fungi are detected for the diagnosis of fungal infections.
45. The method of embodiment 1, wherein in order to treat viral infections, prion-like and/or Tetz-proteins and/or their targets are inactivated.
46. The method of embodiment 1, wherein in order to treat viral infections, prion-like and/or Tetz-proteins are inactivated using specific antibodies against these proteins.
47. The method of embodiment 1, wherein in order to treat viral infections, adjuvants are used that stimulate production of their own specific antibodies inactivating prion-like and/or Tetz-proteins.
48. The method of embodiment 1, wherein in order to treat viral infections, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target.
49. The method of embodiment 1, wherein in order to treat viral infections, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target, while interacting with the target.
50. The method of embodiment 1, wherein the formation of prion-like and/or Tetz-proteins is blocked for the treatment of viral infections.
51. The method of embodiment 1, wherein in order to treat viral infections, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the proteases that lead to their appearance.
52. The method of embodiment 1, wherein in order to treat viral infections, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the extracellular DNA that leads to their appearance alteration of their properties.
53. The method of embodiment 1, wherein in order to treat viral infections, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the ptDNA or ptRNA that leads to their appearance alteration of their properties.
54. The method of embodiment 1, wherein prevention of development of a viral infection is achieved by inactivating the prion-like domains and/or Tetz-proteins on the surface of the viral particles, including those involved in the adsorption and entry.
55. The method of embodiment 1, wherein prevention of development of a viral infection is achieved by inactivating viral prion-like domains and/or Tetz-proteins, including those involved in the biosynthesis, assembly and release of viral particles, as well as those involved in their maturation, inhibition of the virus-induced change in the morphological, biochemical, or growth parameters of a cell, suppression by virus of host complement activation
56. The method of embodiment 1, comprising diagnosis of viral prion proteins and their seeding potential to lead to the formation of misfolded proteins (including, but not limited to protein misfolding cyclic amplification; usage of stains Congo-red, Thioflavin).
57. The method of embodiment 1, comprising diagnosis of Misfolded Aggregates in human biological fluids due to the viral prion proteins (including, but not limited to protein misfolding cyclic amplification; usage of stains Congo-red, Thioflavin).
58. The method of embodiment 1, wherein the evaluation of a presence of viral prion-like domains in microbiota, bodily fluid(s) and/or tissue(s) of the mammal is used as the clinical endpoints in Clinical Trials.
59. The method of embodiment 1, wherein the evaluation of a presence of prion-like and Tetz-proteins and/or the molecules involved in their formation, in microbiota, bodily fluid(s) and/or tissue(s) of the mammal is used as a clinical endpoint in a clinical trial to evaluate treatment efficacy.
60. The method of embodiment 1, comprising diagnostics of the presence of prion-like and Tetz viral proteins and/or component(s) in the blood, plasma or serum of donor and/or recipient during blood during transfusion.
61. The method of embodiment 1, comprising diagnostics of the presence of human host or bacterial host proteins that appear as a result of prion-like and Tetz viral proteins and/or component(s) presence in the blood, plasma or serum of donor and/or recipient during blood during transfusion.
62. The method of embodiment 1, wherein in order to treat bacterial infections, prion-like and/or Tetz-proteins and/or their targets are inactivated.
63. The method of embodiment 1, wherein in order to treat bacterial infections, prion-like and/or Tetz-proteins are inactivated using specific antibodies.
64. The method of embodiment 1, wherein in order to treat bacterial infections, adjuvants are used that stimulate production of their own specific antibodies to inactivate prion-like and/or Tetz-proteins.
65. The method of embodiment 1, wherein in order to treat bacterial infections, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target.
66. The method of embodiment 1, wherein in order to treat bacterial infections, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target, while interacting with the target.
67. The method of embodiment 1, wherein the formation of prion-like and/or Tetz-proteins is blocked in order to treat bacterial infections.
68. The method of embodiment 1, wherein in order to treat bacterial infections, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the proteases that lead to their formation.
69. The method of embodiment 1, wherein in order to treat bacterial infections, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the extracellular DNA that leads to their formation or their properties alterations.
70. The method of embodiment 1, wherein the bacteria producing these prion-like and/or Tetz-proteins are selectively killed for the treatment of bacterial infections.
71. The method of embodiment 1, wherein in order to treat bacterial infections, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the ptDNA or ptRNA that leads to their formation or their properties alterations.
72. The method of embodiment 1, wherein in order to treat infections caused by fungi, prion-like and/or, Tetz-proteins and/or their targets are inactivated.
73. The method of embodiment 1, wherein in order to treat infections caused by fungi, prion-like and/or Tetz-proteins are inactivated using specific antibodies.
74. The method of embodiment 1, wherein in order to treat infections caused by fungi, adjuvants are used that stimulate production of their own specific antibodies inactivating prion-like and/or Tetz-proteins.
75. The method of embodiment 1, wherein in order to treat viral infections caused by fungi, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target.
76. The method of embodiment 1, wherein in order to treat infections caused by fungi, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target, while interacting with the target.
77. The method of embodiment 1, wherein the formation of prion-like and/or Tetz-proteins is blocked in order to treat infections caused by fungi.
78. The method of embodiment 1, wherein in order to treat infections caused by fungi, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the proteases that lead to their appearance.
79. The method of embodiment 1, wherein in order to treat infections caused by fungi, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the extracellular DNA that leads to their appearance alteration of their properties.
80. The method of embodiment 1, wherein the bacteria producing the prion-like and/or Tetz-proteins are selectively killed for the treatment of infections caused by fungi.
81. The method of embodiment 1, wherein in order to treat infections caused by fungi, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the ptDNA or ptRNA that leads to their appearance or alteration of their properties.
82. The method of embodiment 1, wherein in order to treat oncological diseases, prion-like and/or Tetz-proteins are inactivated.
83. The method of embodiment 1, wherein in order to treat oncological diseases, prion-like and/or Tetz-proteins are inactivated using specific antibodies.
84. The method of embodiment 1, wherein in order to treat oncological diseases, adjuvants are used that stimulate production of their own specific antibodies inactivating prion-like and/or Tetz-proteins.
85. The method of embodiment 1, wherein in order to treat oncological diseases, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target.
86. The method of embodiment 1, wherein in order to treat oncological diseases, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target, while interacting with the target.
87. The method of embodiment 1, wherein oncological diseases are treated by prevention of prion-like and/or Tetz-proteins formation.
88. The method of embodiment 1, wherein in order to treat oncological diseases, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the proteases that lead to their appearance.
89. The method of embodiment 1, wherein in order to treat oncological diseases, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the DNA that leads to their appearance alteration of their properties.
90. The method of embodiment 1, wherein in order to treat oncological diseases, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the extracellular DNA that leads to their appearance alteration of their properties.
91. The method of embodiment 1, wherein in order to treat oncological diseases, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the ptDNA or ptRNA that leads to their appearance or alteration of their properties.
92. The method of embodiment 1, wherein in order to treat neurodegenerative and neurodevelopmental diseases, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target.
93. The method of embodiment 1, wherein in order to treat neurodegenerative and neurodevelopmental diseases, prion-like and/or Tetz-proteins are inactivated using molecules that block their interaction with the target, while interacting with the target.
94. The method of embodiment 1, wherein the formation of prion-like and/or Tetz-proteins is blocked for the treatment of neurodegenerative and neurodevelopmental diseases.
95. The method of embodiment 1, wherein in order to treat neurodegenerative and neurodevelopmental diseases, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the proteases that lead to their appearance.
96. The method of embodiment 1, wherein in order to treat neurodegenerative diseases, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the extracellular DNA that leads to their appearance or alteration of their properties.
97. The method of embodiment 1, wherein in order to treat neurodegenerative diseases, the formation of prion-like and/or Tetz-proteins is blocked by inactivating the ptDNA or ptRNA that leads to their appearance or alteration of their properties.
98. The method of embodiment 1, wherein in order to treat neurodegenerative and neurodevelopmental diseases, the effect of viral prion-like domains and/or Tetz-proteins as seed aggregation misfolding proteins is prevented.
99. The method of embodiment 1, wherein in order to generate new synthetic vectors used for gene therapy and gene engineering the number of prion-like domains in viral structures is increased or decreased.
100. The method of embodiment 1, wherein a modified or increased amount of prion-like domains on human cells receptors is used in immunooncology.
101. The method of embodiment 1, wherein modified T-cell ligands with an increased amount of prion-like domains are developed.
102. The method of embodiment 1, wherein modified T-cell ligands with an increased amount of prion-like domains are used for the treatment of mammals.
103. The method of embodiment 1, wherein T-cells with modified ligands containing prion-like domains are used for the treatment of mammals.
104. The method of embodiment 1, wherein T-cells with modified PD1, PD-L1 and CTLA4 containing prion-like domains are used for the treatment of mammals.
105. The method of embodiment 1, wherein modified CAR T-cell technologies with an increased number of prion-like domains to chimeric antigen receptor are used for the treatment of mammals.
106. The method of embodiment 1, wherein modified CAR T-cell technologies with an increased number of prion-like domains to chimeric antigen receptor are used for the treatment of mammals.
107. The method of embodiment 1, wherein the ligand expressed on B cells, plasma cells or plasmablasts in humans is selected from the group consisting of CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, and BCMA CD28, and a binding element for specific interaction with a selected target.
108. The method of embodiment 1, wherein modified CAR T-cell technologies with an increased number of prion-like domains to chimeric antigen receptor is used for the preparation of CAR-T cells with CRISPR/Cas9, and wherein CRISPR/Cas9 CAR is used.
109. The method of embodiment 1, wherein modified CAR T-cell technologies with an increased number of prion-like domains are used to prepare any of the components of an antigen binding domain derived from a bispecific antibody, a transmembrane domain, and a CD3 zeta signaling domain, further wherein the antigen binding domain is selected from the group consisting of a human antibody, a humanized antibody, an antigen binding fragment thereof, and any combination thereof.
110. The method of embodiment 1, wherein modified CAR T-cell technologies with increased number of prion-like domains are used to prepare any of the components of antigen-binding domain, a transmembrane domain, and an intracellular signaling domain or a cytoplasmic co-stimulatory signaling domain.
111. The method of embodiment 1, wherein modified CAR T-cell technologies with increased number of prion-like domains are used to prepare any of the components of antigen-binding domain, a transmembrane domain, and an intracellular signaling domain or a cytoplasmic co-stimulatory signaling domain are used.
112. The method of embodiment 1, wherein modified CAR T-cell technologies with increased number of prion-like domains are developed by an in vitro transcribed RNA or synthetic RNA comprising of a nucleic acid sequence encoding an extracellular domain, a transmembrane domain, a costimulatory signaling region, and/or a signaling domain containing PrD.
113. The method of embodiment 1, wherein synthetic vectors containing an altered amount prion-like and/or Tetz-proteins are used to increase immunogenicity of vaccines.
114. The method of embodiment 1, wherein synthetic vectors containing an altered amount prion-like and/or Tetz-proteins are used to make vaccines.
115. The method of embodiment 1, wherein synthetic vectors containing an altered amount prion-like and/or Tetz-proteins are used to make synthetic vaccines.
116. The method of embodiment 1, wherein synthetic vectors containing an altered amount prion-like and/or Tetz-proteins are used to make recombinant vaccines.
117. The method of embodiment 1, wherein for increasing the activity of vaccines, adjuvants of the vaccines containing increased amount of prion-like and/or Tetz-proteins are used.
118. The method of embodiment 1, wherein for increasing the activity of anticancer vaccines, vaccines containing increased amount of prion-like and/or Tetz-proteins are used.
119. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, the entry of viruses and prokaryotes with prion-like and/or Tetz-proteins into the amniotic fluid is prevented.
120. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, viral and prokaryotic prion-like and/or Tetz-proteins in the amniotic fluid are inactivated.
121. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, viral and prokaryotic prion-like and/or Tetz-proteins in the amniotic fluid which formed under the influence of viral or prokaryotic prion-like and/or proteins Tetz-proteins are inactivated.
122. The method of embodiment 1, wherein in order to treat and prevent congenital mutations and embryogenesis disorders in mammals, entry of viruses and prokaryotes with prion-like and/or Tetz-proteins into the amniotic fluid is prevented.
123. The method of embodiment 1, wherein in order to treat and prevent congenital mutations and embryogenesis disorders in mammals, viral and prokaryotic prion-like and/or Tetz-proteins in the amniotic fluid are inactivated (including by means of antibodies).
124. The method of embodiment 1, wherein in order to treat and prevent congenital mutations and embryogenesis disorders in mammals, viral and prokaryotic prion-like and/or Tetz-proteins in the amniotic fluid which appeared under the influence of viral or prokaryotic prion-like and/or proteins Tetz-proteins are inactivated (including by means of antibodies).
125. The method of embodiment 1, wherein in order to treat and prevent neurodegenerative and neurodevelopmental diseases, the entry of viruses and prokaryotes with prion-like and/or Tetz-proteins into the CSF is prevented.
126. The method of embodiment 1, wherein in order to diagnose diseases in mammals, presence of viral and prokaryotic prion-like and/or Tetz-proteins are detected in CSF.
127. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, viral and prokaryotic prion-like and/or Tetz-proteins are removed or inactivated in the CSF.
128. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, prion-like and/or Tetz-proteins of a mammal which occur under influence of viral and prokaryotic prion-like and/or Tetz-proteins are removed or inactivated in the CSF.
129. The method of embodiment 1, wherein in order to treat and prevent neurodegenerative diseases, viral and prokaryotic prion-like and/or Tetz-proteins are removed or inactivated in the CSF.
130. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, antibodies are used against viral or prokaryotic prion-like and/or Tetz-proteins, and these antibodies are administered to mammals for the purpose of entering the body fluids, including blood and the CSF.
131. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, antibodies are used against prion-like and/or Tetz-proteins of mammals that are formed under influence of viral and prokaryotic prion-like and/or Tetz-proteins are administered to mammals for the purpose of entering the body fluids, including blood and the CSF.
132. The method of embodiment 1, wherein in order to treat and prevent neurodegenerative and neurodevelopmental diseases, antibodies are used against viral or prokaryotic prion-like and/or Tetz-proteins, which are administered to mammals for the purpose of entering the body fluids, including blood and the CSF.
133. The method of embodiment 1, wherein in order to treat and prevent diseases in mammals, antibodies are used against viral or prokaryotic prion-like and/or Tetz-proteins, which are administered to mammals for the purpose of entering the body fluids, including blood and the CSF.
134. The method of embodiment 1, wherein in order to treat and prevent neurodegenerative diseases, antibodies are used against viral or prokaryotic prion-like and/or Tetz-proteins, which are administered to mammals for the purpose of entering the body fluids, including blood and the CSF.
135. The method of embodiment 1, wherein in order to treat and prevent diseases in humans, prion-like and/or Tetz-proteins in the biological fluids are inactivated, which occur as a result of entry of viral and prion-like prokaryotic and/or Tetz-proteins into the human body.
136. The method of embodiment 1, wherein in order to treat and prevent diseases in humans, antibodies against the prion-like and/or Tetz-proteins formed as a result of entry of viral and prion-like prokaryotic and/or Tetz-proteins into the human body are used.
137. The method of embodiment 1, wherein in order to diagnose human diseases, an identification of prion-like domains and viruses carrying prion-like domains is done within biological fluids or mammalian cells.
138. The method of embodiment 1, wherein in order to treat and prevent human diseases, the antiviral action is performed by disrupting the interaction of prion-like domains at the stages of adhesion, entry, biosynthesis, assembly or release, and maturation of viruses.
139. The method of embodiment 1, wherein for preventing and treating viral diseases, in which the prevention of development of a viral infection is achieved by inactivating prion-like domains on the surface of viral particles, including those involved in the adhesion and entry.
140. The method of embodiment 1, wherein for preventing and treating viral diseases, in which the prevention of development of a viral infection is achieved by inactivating viral prion-like domains, including those involved in the biosynthesis, the assembly and release of viral particles, as well as those involved in their maturation, inhibition of virus-induced change in the morphological, biochemical, or growth parameters of a cell, suppression by virus of host complement activation.
141. The method of embodiment 1, wherein for treating and preventing neurodegenerative diseases by preventing effects of viruses as seed aggregation misfolding proteins in the cerebrospinal fluid.
142. The method of embodiment 1, wherein for treating and preventing human diseases by diagnosis of presence of Tetz-proteins and prion-like proteins and PrDs of mammals, prokaryotes and viruses during blood transfusions.
143. The method of embodiment 1, wherein for treating viral infections in mammals by affecting prion-like domains of viruses.
144. The method of embodiment 1, wherein for treating viral infections in mammals by means of antibodies to prion-like domains of viruses.
145. The method of embodiment 1, wherein of treating viral infections of mammals, by means of shared use of antibodies to prion-like domains of viruses together with other drugs.
146. The method of embodiment 1, wherein for increasing the efficiency of antitumoral antibodies by adding prion-like sequences into their amino acid composition.
147. The method of embodiment 1, comprising preparing an antitumoral antibody comprising an amino acid composition of which includes prion-like sequences, wherein the prion-like sequences increase the efficiency of antitumoral antibodies by selection of antibodies to epitopes, antitumoral antibodies.
148. The method of embodiment 1, wherein for increasing the efficiency of oncolytic viruses, viruses are developed with an increased number of prion-like domains or Tetz-proteins on their surfaces.
149. The method of embodiment 1, comprising making synthetic oncolytic viruses by development of viruses with increased number of prion-like domains or Tetz-proteins on their surfaces.
150. The method of embodiment 1, wherein selection of patients entering clinical trials by determining a presence of (i) the prion-like and Tetz-proteins or the molecules involved in their formation present in microbiota, bodily fluid(s) and/or tissue(s) of the mammal. (Monitoring of the prion-like and Tetz-proteins or the molecules involved in their formation, components composition in human body for the Selection of Patients Entering Clinical Trials).
151. A method for diagnosing human diseases by measuring the qualitative and/or quantitative composition of Tetz-proteins prion-like thermostable proteins and mammalian proteins as diagnostic markers.
The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
To identify the PrDs present in viral proteomes, protein sequences were obtained from the UniProt KnowledgeBase (Swiss-Prot and TrEMBL). Protein functions were predicted using the GO terms and manually curated using the information from the UniProt database (UniProt Consortium, “Reorganizing the protein space at the Universal Protein Resource (UniProt)” Nucleic Acids Res., 2012, 40(Database issue): D71-75), the National Center for Biotechnology Information (NCBI), and the literature data (Ashburner et al., 2000).
The presence of PrDs in viral proteomes was analyzed in the known viruses, excluding bacteriophages, using the PLAAC prion prediction algorithm, based on the HMM, and the identification of PrDs was based on the compositional bias towards asparagine and glutamine aminoacyls, an average residue hydrophobicity, and the net charge of sequences. For the analysis the total number of viral proteins contained in the UniProt database was adjusted, since in the proteomes of different viruses, multiple fragments of the same proteins had multiple representation. Therefore, multiple copies of the same sequences were removed in Excel (Windows 10) using the ‘remove duplicates’ function. With the LLR cutoff of 0.003, 2,681 PrDs were identified. The regularities in the likelihood of the identified PrDs to be prions, and their distribution among different viral orders and families were analyzed. The functions of proteins with the identified PrDs were classified using the manually-curated GO categories and were based on the major steps of viral replication. A heatmap was generated using R-statistical computing (see www.r-project.org) with the “levelplot” package. The values in the heatmap range between the lowest and the highest LLR values.
All statistical analyses were conducted using package Statistica for Windows (version 5.0) (StatSoft, Inc.). Data were compared between the viral orders, families, and species by using a χ2 test or the Fisher's exact test. To detect differences in multiple comparisons, one-way analysis of variance (ANOVA) was fitted with the standard confidence interval of 95%. All results were considered statistically significant for p<0.05.
Using the prion-prediction PLAAC algorithm described above, 2,679 PrPs in proteins from 735 different viruses were identified. In total, the inventors analyzed 2,742,160 proteins derived from the UniProtKB database from over 3000 viral species according to the International Committee on Taxonomy of Viruses (ICTV) (Adams et al., 2017).
The average numbers of LLRs varied between the DNA and RNA containing viruses as well as between the enveloped and non-enveloped ones. PrDs were more frequently found in the DNA-containing viruses. Enveloped viruses were also more frequently found to harbor PrD compared with the non-enveloped ones (
High levels of PrDs were found in Herpesvirales, Megavirales, Mononegavirales, Nidovirales, Picornavirales, and Tymovirales (
To analyze the presence of PrDs in different viral orders, the inventors evaluated the ratio between the species identified in this study to possess at least one PrD and the total number of different viral species within that order (Table 1) (Adams et al., 2017).
Herpesvirales
Megavirales
Mononegavirales
Nidovirales
Picornavirales
Tymovirales
The highest number of PrD-containing species are found among Nidovirales and Herpesvirales, with over 93.75% and 71.84% of species, respectively, containing PrDs, while the lowest numbers were found in Tymovirales, with only 8.94% of species with identified PrDs. The inventors have not included the results of Megavirales analysis due to the lack of classification data for this novel viral order (Colson et al., 2013).
Furthermore, the mean number of PrDs per species was calculated as the ratio of the total number of PrDs identified in viral proteomes attributed to an order to the total number of PrD-bearing species identified in this order. The highest average numbers of PrDs per species were identified in Megavirales and Herpesvirales species (Table 3).
Next, the LLRs in the viral orders and families were evaluated. The largest number of viruses with the highest LLR scores, over 50 and 40, were identified in the order Megavirales (families Mimiviridae, Phycodnaviridae, and Poxviridae), while only a few were obtained in Herpesviridae. (Tables 25 and 26). By analyzing top 100 scoring PrDs of the viruses with the greatest prion-forming potential, the inventors evaluated the highest LLR scores predominantly among Megavirales, Herpesviridae, and in viruses of unassigned orders. Twenty seven percent of these top 100 PrDs were identified in the Mimiviridae species, order Megavirales, of Acanthamoeba, with the mean LLR score of 48.68.
Additionally, the PrD enrichment in the proteomes of different viral species was analyzed. The highest enrichment rate was found for the members of the Megavirales order, with at least five PrDs per proteome in the viruses belonging to the Mimiviridae and Phycodnaviridae families. The highest number of different viral species with over 10 PrDs per proteome was found in the Herpesviridae family.
The inventors clustered PrDs into six functional groups based on the major processes during the viral interaction with the host cell: adsorption and entry, biosynthesis, including the transcription, translation, and synthesis of viral components, maturation, assembly, release, and a group comprising proteins with an unknown function (De Clercq, 2002). The inventors separately analyzed the PrDs in the viral precursor proteins (Yost and Marcotrigiano 2013). Additionally, the inventors analyzed the PrDs identified in proteins with the functions not related to the main viral processes, but that, nevertheless, play important roles in disease pathogenesis, the virus-induced changes in the morphological, biochemical, or growth parameters of cells, and the suppression of host complement activation. The correlations the PrDs and protein functions were identified, and the PrD numbers, their LLR scores, and viral families were analyzed (
To facilitate the interpretation of the results, the proteins were grouped based on their functions using the GO terms (
Following this, the inventors identified 433 PrPs (medium LLR score, 5.05) in proteins involved in the viral adsorption and entry, and predominantly associated with the host cell-membrane binding. This group contains proteins belonging to different GO terms, including the integral component of membrane, viral envelope, virion attachment to host cell, fusion of virus membrane with host plasma membrane, receptor-mediated virion attachment to host cell, and others (
The biggest cluster of PrDs (502 proteins) contained the proteins involved in viral transcription, translation, and protein synthesis (LLR score, 6.69), with multiple molecular functions and belonging to different GO terms. The members of Herpesviridae family contained the majority of these PrDs (
Following this, the inventors analyzed PrD-containing viruses associated with the viral assembly. 209 PrDs were identified, with the mean LLR score of 7.79. The main GO terms represented were the viral capsid assembly, serine-type endopeptidase activity, nuclear capsid assembly, viral DNA genome packaging, and others (
The identified PrDs in proteins involved in the release of viral progeny from the host cell were shown to be less abundant, with only 19 proteins found to contain these domains (LLR score, 3.68). In the GO terms, this group predominantly comprised proteins associated with the DNA packaging and viral release from the host cell. The highest number of them were identified in Herpesviridae, including partial proteins, capsid vertex component 2, and tegument protein pp150 (
Additionally, the inventors identified six PrDs in proteins associated with the viral maturation (LLR score, 23.61) and with the GO terms associated with the integral components of the membrane and methyltransferase activity in different viruses (
The PrDs were also detected in 223 structural proteins, predominantly represented by capsid, coat proteins, and hexons (mean LLR score, 5.78) (Ostapchuk and Hearing 2001). Notably, the majority of these proteins was found in different non-enveloped viruses, primarily from the Adenoviridae and Baculoviridae families, and these were less abundant in the enveloped viruses, primarily belonging to Poxviridae (
Furthermore, the inventors identified 138 PrDs (mean LLR score, 6.47) in the viral precursor proteins. Positive-strand RNA viruses are characterized by a positive strand RNA genome encoding a single poly-protein precursor, which, during the post-translational processing, are cleaved and processed into the mature proteins. PrDs were identified in the genome polyproteins of Picornavirales (foot-and-mouth disease virus, enterovirus B, and cardiovirus B) and Flaviviridae (Zika virus, hepatitis C virus), in the Gag polyprotein of Retroviridae (HIV1, bovine leukemia virus), and others (
The PrDs identified in the proteins associated with the viral suppression of host complement activation were less abundant, and only 39 of these proteins were identified (mean LLR score, 7.11). In the GO terms, they were represented with the G-protein coupled receptor activity, evasion or tolerance of host immune response, metal ion binding, and unassigned processes. PrDs were found in NF-kappa B inhibitors, envelope glycoprotein UL33, ankyrin repeat-containing protein, and others, and among different viruses, including some important human pathogens, such as cytomegalovirus, Kaposi's sarcoma-associated herpesvirus, and HIV1 and 2 (Varnum et al., 2004; Chan et al., 2016).
14 PrDs (mean LLR score, 11.09) were found in proteins implicated in the virus-induced change in the morphological, biochemical, or growth parameters of cells. Among these, late membrane protein 1 and K1 were identified in Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus (
Finally, 1097 PrDs were identified in proteins with still unknown functions (mean LLR score, 9.79). The vast majority of these are uncharacterized proteins, which has still not been reviewed in the GO terms.
For probe “N-plasma”, healthy human blood plasma was heated at 100° C. for 5 minutes. For probe “N-plasma+proteinase K”, healthy human blood plasma was incubated for 30 minutes with Proteinase K (100 mcg/ml) followed by heating at 100° C. for 5 minutes. For probe “N-plasma+DNA”, healthy human blood plasma was incubated for 30 minutes with DNA (10 mcg), following by heating at 100° C. for 5 minutes. For, probe “Cancer”, the blood of a patient with breast cancer was heated at 100° C. for 5 minutes. For probe “Cancer+proteinase K”, the blood of a patient with breast cancer was treated with Proteinase K (100 mcg/ml) for 30 minutes and subsequently heated up to 100° C. for 5 minutes.
LC/MS was conducted as previously described. Table 4 below shows a comparison of Tetz-proteins found in the plasma of a healthy volunteers and cancer patients.
Complement C3 (Fragment) OS = GN = C3
11
kDa
13
Serotransferrin (Fragment) OS = GN = TF
8
kDa
9
Gelsolin (Fragment) OS = GN = GSN PE = 1
29
kDa
6
Gelsolin (Fragment) OS = GN = GSN PE = 1
26
kDa
2
Complement C2 OS = GN = C2 PE = 1 SV = 2
83
kDa
4
Complement factor H-related protein 1 OS
31
kDa
6
Pigment epithelium-derived factor OS
46
kDa
8
Cluster of Hemoglobin subunit alpha OS =
15
kDa
5
Hemoglobin subunit alpha OS =
15
kDa
5
CON
—
P01966
?
4
Complement C5 OS = GN = C5 PE = 1 SV = 4
188
kDa
22
Complement C1q subcomponent subunit B (Fragment)
24
kDa
5
Immunoglobulin lambda constant 7 OS =
11
kDa
16
Cluster of Actin, cytoplasmic 1 OS =
42
kDa
12
Actin, cytoplasmic 1 OS = GN = ACTB
42
kDa
12
Coagulation factor XII OS = GN = F12 PE = 1
68
kDa
10
Complement component C6 OS = GN = C6
105
kDa
7
Calmodulin-1 OS = GN = CALM1 PE = 1 SV = 1
17
kDa
7
Thymosin beta-10 OS = GN = TMSB10
5
kDa
2
Cluster of Tropomyosin alpha-4 chain OS =
29
kDa
29
Tropomyosin alpha-4 chain OS = = 3
29
kDa
24
Tropomyosin beta chain OS = GN = TPM2
33
kDa
13
Tropomyosin alpha-4 chain (Fragment)
21
kDa
10
Cluster of Epididymis luminal protein 189
27
kDa
30
Epididymis luminal protein 189
27
kDa
18
Tropomyosin alpha-1 chain
32
kDa
12
Tropomyosin 1 (Alpha), isoform CRA—f
37
kDa
13
Tropomyosin alpha-3 chain
33
kDa
16
Blood plasma of normal healthy volunteers and cancer patients differs in that cancer blood plasma contains certain Tetz-proteins that are not found in normal plasma, and thus can be used for diagnosis. These proteins are indicated in bold in Table 4 and shown below in Table 5.
Some non-limiting examples of such Tetz-proteins include CON_Q2UVX4, Serotransferrin, Gelsolin, Complement C2, Complement factor H-related protein 1, Pigment epithelium-derived factor, Hemoglobin subunit alpha, Complement C5, Complement C1q, Immunoglobulin lambda constant 7, Actin, cytoplasmic 1, Coagulation factor XII, Complement component C6, Calmodulin-1, Tropomyosin alpha-4, Tropomyosin beta Epididymis luminal protein 189, Tropomyosin alpha-1, Tropomyosin alpha-3. Also, certain Tetz-proteins were not found in cancer plasma but were found in plasma for normal healthy volunteers, leading to the altered amount of Tetz-proteins following Tetz-proteins isolation. A non-limiting example includes Complement C4-A. These identified proteins may serve as a qualitative and/or quantitative diagnostic tool. Moreover, the Tetz-proteins whose abundance is changed compared to normal plasma, or which are found solely in cancer specimens, can be used as a target for the treatment.
Table 6 below shows a comparison of Tetz-proteins in the plasma of healthy volunteers, the plasma of healthy volunteers after processing with DNA, and cancer patients. Table 7 below shows the Tetz-proteins that are not present in plasma of healthy volunteers but are present in plasma of healthy volunteers after processing with DNA, and are present in the plasma of patients with cancer.
CON
—
Q2UVX4
7
7
Serotransferrin (Fragment) OS = GN = TF
5
9
Complement factor H-related protein 1 OS =
4
6
Pigment epithelium-derived factor OS = 4
3
8
Cluster of Hemoglobin subunit alpha)
5
5
Hemoglobin subunit alpha
5
5
CON
—
P01966
4
4
Complement C5 OS = GN = C5 PE = 1 SV = 4
6
22
Immunoglobulin lambda constant 7
12
16
Homo
Homo sapiens
Treatment of blood plasma with DNA led to the (a) formation of certain Tetz-proteins that are not found in normal plasma, but are typical/found in cancer specimens. These proteins are indicated in bold in Table 6 and shown above in Table 7. Non-limiting examples include: CON_Q2UVX4, Serotransferrin, Complement factor H-related protein 1, Pigment epithelium-derived factor, Cluster of Hemoglobin subunit alpha, Hemoglobin subunit alpha, CON_P01966, Complement C5, Immunoglobulin lambda constant 7. Also, treatment with DNA altered the amount of Tetz-proteins in a normal sample such that the amount of Tetz-proteins was similar to that found in cancer samples. Therefore, the addition of DNA (including, but not limited to eukaryotic, prokaryotic or extracellular prokaryotic DNA) to the blood specimens can be used for the diagnostics of human diseases. Moreover, increased amounts of bacterial DNA in blood plasma can lead to the formation of altered Tetz-proteins and thus can be used a therapeutic target.
Table 8 below shows a comparison of Tetz-proteins in plasma of healthy volunteers and cancer patients, both before and after processing with proteases.
Treatment of blood plasma of volunteers and cancer patients with proteases led to the (a) formation of certain Tetz-proteins that are not found in normal plasma but are found in cancer blood plasma. Exemplary such proteins are listed in Table 9.
Such Tetz-proteins can be used for diagnosis, with non-limiting examples including Complement C3, CON_Q2UVX4, Serotransferrin, Gelsolin, Immunoglobulin lambda constant 7, and Inter-alpha-trypsin inhibitor heavy chain H3. Other Tetz-proteins form in cancer blood plasma with protease treatment that are not formed in normal plasma. These Tetz-proteins can be used for diagnosis. Examples of such proteins are listed in Table 10. Treatment of blood plasma of cancer patients with proteases can lead to decreased levels of a set of Tetz-proteins (for example, those listed in Table 11) while treatment of blood plasma of healthy subjects (or volunteers) either increases or does not significantly change the levels of the Tetz-proteins in the same set.
Further, the protease activity that leads to the formation of novel/altered abundance of Tetz-proteins in cancer patients can be used as a therapeutic target.
5 ml of blood plasma of healthy volunteers was used and was divided into the groups listed below. Each group was heated with different temperature regimens and/or treated with proteinase K (Sigma Aldrich) from 10 to 250 mcg/ml and/or treated with DNA from 10 to 250 mcg/ml:
Group #1—plasma heated at 80° C. for 1 minute
Group #2—plasma heated at 80° C. for 30 minutes
Group #3—plasma heated at 100° C. for 15 minutes
Group #4—plasma heated at 150° C. for 15 minutes
Group #5—plasma heated at 100° C. for 15 min treated with proteinase K 10 mcg/ml for 30 min (37° C.)
Group #6—plasma heated at 100° C. for 15 minutes+treated with proteinase K 10 mcg/ml for 30 min at room temperature
Group #7—plasma heated at 100° C. for 15 minutes+treated with proteinase K 250 mcg/ml for 30 min (37° C.)
Group #8—plasma heated at 100° C. for 15 minutes+treated with proteinase K 100 mcg/ml for 3 min at room temperature
Group #9—plasma heated at 100° C. for 15 minutes+treated with DNA 100 mcg/ml for 30 min at 37° C.
Group #10—plasma heated at 100° C. for 15 minutes+treated with DNA 1 mcg/ml for 30 min at 37° C.
Group #11—plasma heated at 100° C. for 15 minutes+treated with DNA 1 mcg/ml for 30 min at 37° C. and treated with proteinase K 100 mcg/ml for 30 min at 37° C.
Protein bands were analyzed with gel electrophoresis and subsequently subjected to LC/MS analysis. Electrophoresis was conducted in the BIO-RAD Mini PROTEAN Tetra Cell (Bio-Rad Laboratories) at 60 volts for about 40 minutes with 12% polyacrylamide gel (Bio-Rad Laboratories). Proteins were stained with Coomassie blue and then were destained with Destining Solution according to manufacturers instructions (all Bio-Rad Laboratories).
LC/MS analysis was conducted using nanoflow UPLC-MS/MS (Thermo Q Exactive HF Orbitrap) in which ultra high-performance liquid chromatography was coupled to tandem mass spectrometry according to the manufacturer's instructions.
Analysis for the presence of prion-like domains in the identified proteins was conducted with prion-like amino acid composition algorithm (PLAAC) (plaac.wi.mit.edu). PLAAC analysis involves evaluation of proteins to determine if they contain prion-like domains, defined as domains with compositional similarity to yeast prion domains, based on amino-acid interaction sets (Michelitsch and Weissman, 2000; Bathe et al., 2017). The resulting log-likelihood ratio (LLR) indicates the possibility that the analyzed protein is a prion. Using PLAAC algorithms, PrDs, defined as domains shown to contain at least a domain compositionally similar to yeast prions, were recently investigated in different species, both eukaryotic and prokaryotic, confirming their important regulatory and functional roles (Malinovska et al., 2013; Iglesias et al., 2015; March et al., 2016; Tetz and Tetz 2017).
The presence of thermostable proteins was found in all the groups. Moreover, the alteration of the electrophoretic profile of these proteins under proteinase K and DNA treatment was identical for all the probes. The results for some of them are shown in
Thermostable proteins from healthy blood plasma before and after proteinase K and DNA treatment lack known prion-like domains and the amounts of such proteins were changed by processing with proteases, nucleic acids, or combinations of proteases and nucleic acids. Identified thermostable proteins were analyzed with a PLAAC algorithm dedicated to identify prion-like domains that could address thermostable properties of these proteins, but have not found any prion-like domains within these proteins (for some proteins data are illustrated with
5 ml of cerebrospinal fluid (CSF) of healthy volunteers was used and divided into the following groups. Each group was heated with different temperature regimens and/or treated with proteinase K (Sigma Aldrich) from 10 to 250 mcg/ml and/or treated with DNA from 10 to 250 mcg/ml:
Group #1—CSF heated at 80° C. for 1 minute
Group #2—CSF heated at 80° C. for 30 minutes
Group #3—CSF heated at 100° C. for 15 minutes
Group #4—CSF heated at 150° C. for 15 minutes
Group #5—CSF heated at 100° C. for 15 minutes+treated with proteinase K 10 mcg/ml for 30 min at 37° C.
Group #6—CSF heated at 100° C. for 15 minutes+treated with proteinase K 10 mcg/ml for 30 min at room temperature
Group #7—CSF heated at 100° C. for 15 minutes+treated with proteinase K 250 mcg/ml for 30 min at 37° C.
Group #8—CSF heated at 100° C. for 15 minutes+treated with proteinase K 100 mcg/ml for 3 min at room temperature
Group #9—CSF heated at 100° C. for 15 minutes+treated with DNA 100 mcg/ml for 30 min at 37° C.
Group #10—CSF heated at 100° C. for 15 minutes+treated with DNA 1 mcg/ml for 30 min at 37° C.
Group #11—CSF heated at 100° C. for 15 minutes+treated with DNA 1 mcg/ml for 30 min at 37° C. and treated with proteinase K 100 mcg/ml for 30 min at 37° C.
Protein bands were analyzed with gel electrophoresis and subsequent LC/MS analysis. Electrophoresis was conducted in the BIO-RAD Mini PROTEAN Tetra Cell (Bio-Rad Laboratories) at 60 volts for about 40 minutes with 12% polyacrylamide gel (Bio-Rad Laboratories). Proteins were stained with Coomassie blue and then were destained with Destaining Solution according to the manufacturers instructions (all Bio-Rad Laboratories).
LC/MS analysis was conducted using nanoflow UPLC-MS/MS (Thermo Q Exactive HF Orbitrap)—that is, ultra high-performance liquid chromatography coupled to tandem mass spectrometry according to the manufacturer's instruction.
Analysis for the presence of prion-like domains in the identified proteins was conducted with the prion-like amino acid composition algorithm (PLAAC) (plaac.wi.mit.edu). PLAAC analysis, which allows the evaluation proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, based on amino-acid interaction sets (Michelitsch and Weissman, 2000; Bathe et al., 2017). The resulting log-likelihood ratio (LLR) indicates the possibility that the analyzed protein is a prion. Using PLAAC algorithms, PrDs, defined as domains shown to contain at least a domain compositionally similar to yeast prions, were recently investigated in different species, both eukaryotic and prokaryotic, confirming their important regulatory and functional roles (Malinovska et al., 2013; Iglesias et al., 2015; March et al., 2016; Tetz and Tetz 2017).
The presence of thermostable proteins was found in all the groups. Moreover, the alteration of the electrophoretic profile of these proteins under proteinase K and DNA treatment was identical for all the probes. The results for some of them are shown in
As it is seen, CSF possesses thermostable proteins. The content of these proteins is changed under Proteinase K treatment.
Table 12 demonstrates thermostable proteins from healthy CSF before and after proteinase K treatment. Identified thermostable proteins were analyzed with a PLAAC algorithm dedicated to identifying prion-like domains, that could address thermostable properties of these proteins. The inventors did not find any prion-like domains within these proteins (for some proteins data are illustrated with
To identify the PrDs present in viral proteomes, protein sequences were obtained from the UniProt KnowledgeBase (Swiss-Prot and TrEMBL). The presence of PrDs in viral proteomes was analyzed in the known viruses, excluding bacteriophages, using the PLAAC prion prediction algorithm, based on the HMM, and the identification of PrDs was based on the compositional bias towards asparagine and glutamine aminoacyls, an average residue hydrophobicity, and the net charge of sequences. For the analysis the total number of viral proteins was contained in the UniProt database was adjusted, since in the proteomes of different viruses, multiple fragments of the same proteins had multiple representation. Therefore, multiple copies of the same sequences were removed in Excel (Windows 10) using the ‘remove duplicates’ function. With the LLR cutoff of 0.003, 2,681 PrDs were identified. The regularities in the likelihood of the identified PrDs to be prions, and their distribution among different viral orders and families were analyzed. The functions of proteins with the identified PrDs were classified using the manually-curated GO categories and were based on the major steps of viral replication. A heatmap was generated using R-statistical computing (www.r-project.org) with the “levelplot” package. The values in the heatmap range between the lowest and the highest LLR values.
A list of viral species in which at least one prion-like domain was identified is found in Table 13.
Choristoneura murinana nucleopolyhedrovirus
A list of prion-like domains in human Herpes Virus 1 is shown in Table 15 below.
An anti-PrD drug called Tacrolimus was used to study its possible antiviral activity. Tacrolimus is an anti-PrD drug with known activity against prions but is not known to have antiviral activity. For anti-HSV activity, Vero cells were seeded in 24-well plates at a density of 70×103 cells. After 24 h, the cells were treated with a clinical isolate of HSV-1 at a multiplicity of infection (MOI) of 0.1 PFU/cell. Following virus adsorption (2 h at 37° C.), Tacrolimus was added and cultures were maintained in medium containing for another 48 h until control cultures displayed extensive cytopathology. It has been thus determined that Tacrolimus has the antiviral activity against the herpes virus used (type I), as seen in Table 16 below.
As can be seen from the data presented, an anti-PrD drug without a known antiviral activity inhibits the reproduction of herpesviruses which proteome is enriched in prion-like domains. Thus, an antiprionogenic drug possesses antiviral activity against prion-containing viruses.
The effect of anti-PrD drugs on biofilm amyloid formation was analyzed using a Congo Red assay. It is known that when Congo Red (CR) interacts with microbial amyloid, it also produces a bright red fluorescence that can be quantified with an excitation wavelength of 485 nm and an emission wavelength of 612 nm (Zhou, Yizhou, et al. “Bacterial amyloids.” Amyloid Proteins. Humana Press, 2012. 303-320.)
Tacrolimus, Pentosan polysulfate, and Quinacrine were used as drugs with known anti-prion activity (Karapetyan, Yervand Eduard, et al. “Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents.” Proceedings of the National Academy of Sciences 110.17 (2013): 7044-7049.; Rahman, Ziyaur, Ahmed Zidan, and Mansoor A. Khan. “Tacrolimus properties and formulations: potential impact of product quality on safety and efficacy.” Tacrolimus: Effectiveness, Safety and Drug Interactions, Nova Science Publishers Inc., New York (2013): 1-39.; Farquhar, C., Dickinson, A., & Bruce, M. (1999). Prophylactic potential of pentosan polysulphate in transmissible spongiform encephalopathies. The Lancet, 353(9147), 117.; Geschwind, Michael D., et al. “Quinacrine treatment trial for sporadic Creutzfeldt-Jakob disease.” Neurology 81.23 (2013): 2015-2023.; Geschwind, M. D.; Kuo, A.; Raudabaugh, B.; Haman, A.; Devereux G.; Johnson D. Y.; Torres-Chae, C.; Wong K. S.; Prusiner S.; Miller B. L. The first U.S. treatment trial for sporadic CJD. In: Abstracts of the 134th Annual Meeting of The American Neurological Association. Oct. 11-14, 2009. Baltimore, Md., USA. Ann Neurol., 2009, 66, S49-S50 (Abs).)
Inhibition of Congo-red and inhibition of biofilm formation was examined by directly applying the anti-PrD drugs at time zero to a growing culture in liquid medium at 37° C. Cells were analyzed for CR binding when reached absorbance (A600 nm) of approximately 1.2 absorbance units (AU). The amyloid-producing bacteria displayed elevated levels of CR binding. However, in the presence of the Tacrolimus, Pentosan polysulfate or Quinacrine, about 40% decrease in CR binding was observed, suggesting that the drugs affect amyloid production.
A biofilm formation assay was undertaken. An inoculum of amyloid producing Escherichia coli strain VT-156 and non-amyloid producer Escherichia coli strain RA-74 were prepared by using a 24-h broth culture. The inoculum, which contained 7.53+/−0.22 log 10 CFU/ml, was added to the wells of 96-well plates (200 mcl/well), 35-mm petri dishes (2 ml), and coverslips that were placed in glass tubes (2 ml) (all from Sarstedt, Germany); and the plates, dishes, and coverslips were incubated at 37° C. for 24 h. The effect of the anti-PrD drugs on a 24 hour old S. aureus biofilm (beta amyloid formation) is shown in Table 17 below.
Escherichia coli
Escherichia coli
As can be seen from the data presented, the anti-PrD drugs inhibited formation of bacterial amyloid (based on CR assay) and inhibited biofilm formation of amyloid-producing bacteria of microbial biofilms. Thus, these drugs possess antimicrobial and antibiofilm activity.
The effect on the model of increased gut permeability in mice was studied. Increased gut permeability allows increased levels of bacterial DNA in the blood circulation, leading to the increase in the level of beta amyloid (Bala, S. et al., 2014; DiBiagio, J. R. et al., 2016). DNAse prevents the appearance of a thermostable protein formed by the extracellular DNA.
Hemizygous transgenic mice expressing familial Alzheimer's disease mutant human (line Tg2576, Hsiao et al., 1996). A total of 70 male mice were used. To identify beta-amyloid (Aβ) amount, one quarter brain from each animal was homogenized in 70% formic acid at a weight:volume ratio of 100 mg/ml. The homogenate was sonicated for 2 min and centrifuged at 100,000 g for 1 h. After centrifugation, the supernatant fraction was removed and neutralized with 19 vol of Tris-phosphate buffer. Samples were analyzed by a modified sandwich ELISA that detects total Aβ. To detect human Aβ, aliquots of homogenate were added to Nunc Maxisorb plates coated with monoclonal antibody 6E10 (Senetek) capture antibody. After incubation at 4° C. overnight, human Ab was detected by monoclonal antibody 4G8 (Senetek) conjugated to horseradish peroxidase (HRP). After washing with PBS containing 0.05% Tween 20, the bound peroxidase was detected by the TMB peroxidase kit (Kirkegaard & Perry). Plates were read at 450 nm in a standard plate reader, and unknowns were quantified by comparison to known quantities of freshly dissolved Ab40 (Bachem).
Animals were injected daily, with increasing concentrations of S. aureus bacterial DNA. Control animals were injected with sterile water. DNase (2000 Kunitz units) was administered orally or by IV on day 1. Data are presented in Table 18.
As it is seen, inactivation of DNA lead to the decreased amount of amyloid-beta in the mice brains. Thus, the destruction of extracellular DNA can be used for the prevention of prion-like and/or Tetz-proteins formation.
The effect of antibodies against bacterial amyloid on amyloid formation was analyzed using Congo red assay. It is known that when Congo red (CR) interacts with microbial amyloid, it also produces a bright red fluorescence that can be quantified with an excitation wavelength of 485 nm and an emission wavelength of 612 nm (Zhou, Yizhou, et al. “Bacterial amyloids.” Amyloid Proteins, Humana Press, 2012. 303-320.)
Antibodies were obtained by immunizing the rabbit by alfa arnyloid. Amyloid was received from bacterial biofilm. Chai, L. et al., “Isolation, characterization, and aggregation of a structured bacterial matrix precursor” J. Biol. Chem. 2013 Jun. 14; 288(24):17559-68.
Inhibition of Congo-red and inhibition of biofilm formation were examined by directly applying the anti-PrD drugs at time zero to a growing culture in liquid medium at 37° C. Cells were analyzed for CR binding when reached absorbance (A600 nm) of approximately 1.2 absorbance units (AU). The amyloid-producing bacteria displayed elevated levels of CR binding. However, in the presence of the antibodies to amyloid about 30% decrease in CR binding was observed, suggesting that the drugs affect amyloid production. Table 19 below shows amyloid beta formation in a biofilm of E. coli.
Biofilm formation assay. An inoculum of amyloid producing Escherichia coli strain VT-156 and an inoculum of non-amyloid producer Escherichia coli strain RA-74 were prepared by using a 24-h broth culture. Each inoculum, which contained 7.53+/−0.22 log 10 CFU/ml, was added to the wells of 96-well plates (200 mcl/well) and 35-mm petri dishes (2 ml). Coverslips were placed in glass tubes (2 ml) (all from Sarstedt, Germany); and the plates, dishes, and coverslips were incubated at 37° C. for 24 h. Table 20 below shows the effect on S. aureus biofilm beta amyloid formation over 24 hours.
Escherichia coli
Escherichia coli
As can be seen from the data presented, the antibodies against amyloid inhibited formation of bacterial amyloid (based on CR assay) and inhibited biofilm formation of amyloid-producing bacteria of microbial biofilms, thus possessing antimicrobial and antibiofilm activity.
Antibodies were obtained by immunizing a rabbit by alfa amyloid. Amyloid was received from S. aureus VT-177 bacterial biofilm. Chai, L. et al., “Isolation, characterization, and aggregation of a structured bacterial matrix precursor” J. Biol. Chem. 2013 Jun. 14; 288(24):17559-68.
C57B1 mice and white randomly bred mice were used. The weight of animals was 24-26 g. 6-7 animals were kept in one cage on a standard diet without limitation of water. Animals were administered amyloid of S. aureus VT-177, IV, 10 mcg/mL for 6 days. The experimental group was administered IV antibodies starting from day 1. Control animals were administered IV sterile water. Each group contained 10 animals. Animal survival was measured as a primary endpoint. Table 21 below shows the number of animals that died in each of the groups of 10 animals.
The data obtained revealed that antibodies against bacterial amyloid results in significant protection from bacterial-related mortality.
Antibodies were obtained by immunizing the rabbit by Proteinase K and can be used for both diagnostics and treatment of human malignancies. It is known that human blood plasma and CSF in cancer patients, and patients neurodegenerative and autoimmune diseases, possess elevated levels of proteases (Tamkovich, Svetlana, and Olga Bryzgunova. “Protease Activity and Cell-Free DNA in Blood Plasma of Healthy Donors and Breast Cancer Patients.” Journal of Immunoassay and Immunochemistry, 37.2 (2016): 141-153; Andreasson, Ulf, et al. “An enzyme activity as a potential biomarker for Alzheimer's disease.” Alzheimer's & Dementia: The Journal of the Alzheimer's Association, 6.4 (2010): S497-S498.). Thus, inactivation of proteases is suggested to have therapeutic potential.
To the human blood plasma, antibodies to proteinase K were added. After a 30-minute exposure, the plasma was heated in a boiling water bath for 15 minutes. The liquid fraction was separated further and studied by electrophoresis in 12% gel. (
Identification of Tetz-proteins can be suggested as a novel diagnostic criteria for cancer diagnostics.
As it is seen, identification of Tetz-proteins allows detection of an altered amounts of proteins and the appearance of unique proteins that can be visualized by different methods including SDS electrophoresis (red arrows). Processing with proteases leads to the formation of altered proteins, and unique proteins that could be detected and used for the diagnosis of oncological diseases (black arrows).
Identification of Tetz-proteins may be a novel diagnostic criteria for neurodegenerative diseases.
As it is seen, identification of Tetz-proteins allows detection of an altered amounts of proteins and the appearance of unique proteins in neurodegenerative diseases that can be visualized by different methods including SDS electrophoresis (red arrows). Processing with proteases lead to the formation of altered proteins, and unique proteins that could be detected and used for the diagnosis of neurodegenerative diseases (black arrows).
LC/MS was conducted as previously described. The mass spectrometry data for a (Probe_1) healthy human blood plasma heated for 100° C. for 5 minutes; (probe_2) healthy human blood plasma after incubation for 30 minutes with Proteinase K, following heating for 100° C. for 5 minutes; (Probe_3) blood of a patient with breast cancer heated for 100° C. for 5 minutes (probe_4) blood of a patient with breast cancer treated with Proteinase K for 30 minutes and subsequent heating up to 100° C. for 15 minutes.
There was difference in the Tetz-proteins and prion-like proteins abundance, with and without proteinases treatment. Key alternations are presented in Table 22 below, indicating altered abundance [marked with underlining] and appearance of novel proteins [marked in bold], of blood Tetz and prion-like proteins in human blood plasma with and without proteinase treatment.
729
729
676
186
186
13
7
121
121
9
94
94
82
65
59
37
36
35
51
48
6
39
29
23
30
39
39
28
24
26
20
24
10
40
13
16
12
12
2
2
7
2
1
1
4
10
10
7
7
3
2
1
2
2
2
1
1
2
1
1
2
117
5
13
17
4
2
2
4
2
2
9
3
4
29
24
13
10
30
18
12
13
16
3
4
3
2
2
4
3
5
2
1
6
2
3
2
2
2
5
3
3
1
4
3
2
2
2
3
3
3
2
2
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
As can be seen from the presented data above, this method allows to identify the difference in the representation of Tetz-proteins in patients with diseases, including by means of addition of proteinases. Moreover, certain proteins following protease treatment had different trends in the alteration of their amount. Thus, for example CO3_HUMAN was increased following proteinase procession of normal blood plasma, but was decreased in cancer patients.
The electrophoretic profile of thermostable blood plasma of patients was analyzed. Blood plasma specimens were heated for 10 minutes in a water bath. Electrophoresis of proteins was performed by the Bio-Rad system according to the instructions of the manufacturer [www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf].
The electrophoretic profile of thermostable and proteinase-resistant blood plasma of patients were analyzed. Blood plasma specimens were mixed with proteinase K (100 mcg/ml) for 30 minutes and then heated for 10 minutes at water bath. Electrophoresis of proteins was performed by the Bio-Rad system according to the instructions of the manufacturer [www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf].
The diagnostics of mammalian diseases, using alteration of the proteomic content of biological fluids following DNA processing. Erlich carcinoma was modelled in mice. Cells were cultivated in RPMI-1640 medium with 10% calf serum and 1% penicillin-streptomycin in an atmosphere of 5% CO2. For tumor inoculation in mice, the cells were cultivated till monolayer is formed, then detached with trypsin-TA buffer. The cells were washed 3 times by centrifuging in phosphate buffer and then resuspended up to 0.5×107/ml concentration in the same buffer. The cell viability was determined with methylene blue staining in a hemocytometer. Cells suspensions with no less than 95% of living cell were used for transplantation.
C57B1 mice and white randomly bred mice were used. The weight of the animals was 24-26 g. 6-7 animals were kept in one cage on a standard diet without limitation of water. Erlich tumors were transplanted by administration of 0.2 ml of 10% cell suspension in physiological solution.
Blood plasma was taken before the initiation of cancer and after. To the plasma probes ex vivo, DNA was added (5 mcg). The electrophoretic profiles of thermostable and proteinase-resistant blood plasma were analyzed. Blood plasma specimens were pre-incubated with DNA for 30 minutes at 37° C., then mixed with proteinase K (100 mcg/ml) for 30 minutes and then heated for 10 minutes at water bath. Electrophoresis of proteins was performed by the Bio-Rad system according to the instructions of the manufacturer [www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf].
Erlich carcinoma was modelled in mice. Cells were cultivated in RPMI-1640 medium with 10% calf serum and 1% penicillin-streptomycin in atmosphere of 5% CO2. For tumor inoculation in mice, the cells were cultivated until a monolayer is formed and then were detached with trypsin-TA buffer. The cells were washed 3 times by centrifuging in phosphate buffer and then resuspended up to 0.5×107/ml concentration in the same buffer. The cell viability was determined with methylene blue staining in a hemocytometer. Cells suspensions with no less than 95% of living cells were used for transplantation.
C57B1 mice and white randomly bred mice were used. Weight of animals was 24-26 g. 6-7 animals were kept in one cage on a standard diet without limitation of water. Erlich tumors were transplanted by administration of 0.2 ml of 10% cell suspension in physiological solution.
Blood plasma was taken before the initiation of cancer and after.
Resulting antibodies were given (IV) in the same animal model 7 days after cancer initiation.
The mortality rate was analyzed for 45 days. Table 23 shows the mortality rate on the 45th day of the experiment.
As it is seen, the destruction/inactivation of Tetz-proteins and prion-like proteins can be used for the treatment of cancer.
AAV5x2 and AAV5x5 vectors were synthetically constructed from AAV5 that had 2 and 5 times more PrDs (capsid proteins VP1 with Prion-like-domain) on their surfaces.
To identify the PrDs present in viral proteomes, protein sequences were obtained from the UniProt KnowledgeBase (Swiss-Prot and TrEMBL). The presence of PrDs in viral proteomes was analyzed, using the PLAAC prion prediction algorithm, based on the HMM, and the identification of PrDs was based on the compositional bias towards asparagine and glutamine aminoacyls, an average residue hydrophobicity, and the net charge of sequences. The data of
Adult male mice C57Bl/6 (20-22 g) were housed at an ambient temperature of 21° C. with a 12:12 hour light-dark cycle. Food was provided ad libidum, as was water. The adeno-associated virus—5 (AAV) was serotype 5, with a transgene cassette containing the promoter driving expression of PrDs. Viral stock was purified by CsCl step and isopycnic gradient centrifugation. The vector was then dialyzed into 50% glycerol as a cryoprotective in a buffer (10 mM Tris, 10 mM His, 75 mM NaCl, 0.5% v/v EtOH, 1 mM MgCl, 0.1 mM EDTA, and 50% v/v glycerol) optimized for the maintenance of adenoviral viability, and diluted in PBS immediately prior to the injection. The concentration of the highly purified virus was determined spectrophotometrically, with one OD260 equivalent to 1012 particles/ml and a particle:pfu ratio of 100:1. Vectors were injected IV 5×1011 gc/mouse. Biodistribution (C57Bl/6) was measured. The results are shown in Table 24 below.
An increase in the representation of PrDs can allow for increased organ-specific expression.
In total, 30 samples of Kaposi's sarcoma from four patients were studied. Fixed, paraffin-embedded tissue sections were then examined immunohistochemically using the monoclonal antibody to glycoprotein gp160 or to the PrDs region of glycoprotein gp160 of Human Herpes Virus 8. Rat monoclonal antibodies to HHV-8 LNA-1, ORF73 (Advanced Biotechnologies Inc.) were used as a positive control.
Experimental antibodies were developed by immunizing the rabbit by PrDs part of Envelope glycoprotein gp160 of Human Herpes Virus 8. Antibodies were obtained by immunizing the rabbit by gp160 or by PrDs part of gp160. The amino acid sequence of Envelope glycoprotein gp160 Human Herpes Virus 8 is
The PrD of Envelope glycoprotein GP4 Human Herpes Virus 3 is shown in
Antibodies that recognize the HHV-3 gH/gL protein complex were used as a positive control. Surviving virus was titrated on subconfluent HFL monolayers propagated in 1 ml of 10% FBS-DMEM in 12-well plates. Plaque reduction is expressed in percent virus survival for triplicate experiments. The data is shown in
As it can be seen the concentration of antibodies to gH/gL that produced 50% plaque reduction was 0.6 μg/ml, while antibodies to GP4 and antibodies to PrD GP4 that produced 50% plaque reduction were 0.1 μg/ml and 0.06 μg/ml respectively.
Tissue sections were stained and were used to detect rat antibodies (three drops rabbit normal serum concentrate, one drop biotinylated rabbit, anti-rat secondary antibody (Vector Laboratories, Burlingame, Calif., USA) for every 10 ml of biotinylated immunoglobulin from the standard DAB detection kit). Primary antibody dilution was 1:1000 with an incubation time of 30 min. Antigen retrieval was achieved with a 15-min treatment in a microwave pressure cooker with citrate buffer, followed by a 15-min cool down. A cell block of the primary effusion lymphoma cell line BC-3 and a sample from a patient having Kaposi's sarcoma were used as positive controls. The results are shown in Table 25 below.
The above data indicate that the use of antibodies to proteins with PrDs and antibodies to the epitopes containing PrDs increases sensitivity of diagnostics. The properties of prion-like proteins allow the use of antibodies in a wide range of diagnostic methods, including, but not limited to Western Blot or monoclonal antibody-blocking EIA, enzyme-linked immunosorbent assay (ELISA), and others.
Human lung fibroblast (HFL) cells (ATCC, VA) were cultured in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine (DMEM; Sigma-Aldrich, St. Louis, Mo.) and 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, Ga.). Human Herpes Virus 3 (HHV-3) was propagated by co-cultivating infected cells with uninfected cell. Infected HFL cultures were harvested at the height of virus-induced cytopathic effect, for 72 h postinfection (dpi). To study the plaque reduction antibody neutralization assay, aliquots of VZV-infected HFL cells were incubated for 60 min at 37° C. in 50 μl DMEM containing increasing amounts of monoclonal antibodies. The inventors developed antibodies to GP4 protein or to the PrD of GP4 protein. The amino acid sequence of the Envelope GP4 Human Herpes Virus 3 is
The PrD of Envelope glycoprotein GP4 Human Herpes Virus 3 is shown in
Antibodies that recognize the HHV-3 gH/gL protein complex were used as a positive control. Surviving virus was titrated on subconfluent HFL monolayers propagated in 1 ml of 10% FBS-DMEM in 12-well plates. Plaque reduction is expressed as percent virus survival for triplicate experiments. The data are shown in
As it can be seen the concentration of antibodies to gH/gL that produced 50% plaque reduction was 0.01 μg/ml, while antibodies to GP4 and antibodies to PrD GP4 that produced 50% plaque reduction were 0.6 μg/ml and 0.06 μg/ml respectively.
Modified Rituximab antibodies (Rituximab-Mod) with added PrDs were constructed. Rituximab, in contrast, has no PrDs.
The sequence of the heavy chain of the Rituximab chimeric antibody is as follows.
The PrD of the heavy chain of the Rituximab chimeric antibody is shown in
The sequence of the light chain of the Rituximab chimeric antibody is as follows.
The PrD of the light chain of the Rituximab chimeric antibody is shown in
Rituximab-Mod has PrD. The sequence of Rituximab-Mod is shown below, with the PrDs underlined and in bold.
The PrD of the heavy chain of Rituximab-Mod is shown in
The efficacies of Rituximab and Rituximab-Mod were compared based on Binding to CD20-expressing target cells and antibody-dependent cell-mediated cytotoxicity [ADCC] assays.
The binding of Rituximab-Mod and rituximab was assessed on SU-DHL4 cell lines with high (1,000,000) CD20 receptor copy numbers. Titration of antibody concentrations up to 10 μg/mL showed that the maximal binding intensity of Rituximab-Mod to tumor cells was over 2 times that observed with the same concentrations of rituximab. The ability of Rituximab-Mod and rituximab to mediate ADCC was assessed using SU-DHL4 target cell lines. The potency of Rituximab-Mod was higher than that of rituximab. The superiority of Rituximab-Mod was apparent in terms of both EC50 values of target cell killing (˜0.1 ng/mL for Rituximab-Mod vs. ˜10 ng/mL for rituximab) and higher overall killing efficacy, particularly at low antibody concentrations.
5 ml of blood plasma of a patient with breast cancer was used and heated at 100° C. for 5 minutes. Proteins were analyzed with LC/MS analysis, which was conducted using nanoflow UPLC-MS/MS (Thermo Q Exactive HF Orbitrap) in which ultra high-performance liquid chromatography was coupled to tandem mass spectrometry according to the manufacturer's instructions.
The presence of thermostable proteins and their parts was found. Almost all of the identified proteins are known to be associated with different cancers.
Non-limiting examples of identified thermostable Tetz-proteins are shown in Table 26 below.
Cancer letters 121.2 (1997): 139-145.
and Prevention Biomarkers (2015).
Research 4.10 (1998): 2511-2520.
Identification of Tetz-proteins allows for simultaneous evaluation of a large variety of different cancer biomarkers. Such simultaneous analysis can be useful for the diagnosing neoplastic processes. Analysis of a correlation between Tetz-proteins that are also known as oncomarkers and/or other Tetz-proteins can be used for the development of algorithms for the specific evaluation of certain cancers.
0.5 ml of blood plasma of control patient with no known oncology and 0.5 ml of blood plasma of patient with breast cancer were used and heated at 100° C. for 5 minutes. Proteins were analyzed with LC/MS analysis, which was conducted using nanoflow UPLC-MS/MS (Thermo Q Exactive HF Orbitrap) in which ultra high-performance liquid chromatography was coupled to tandem mass spectrometry according to the manufacturer's instructions.
The presence of thermostable proteins and their parts was found in both the groups. Moreover, among these proteins there was a large number of cancer biomarkers, that are known to be associated with different cancers.
The effects of added DNA, added proteinase K and added DNA plus proteinase K are shown in a set of thermostable proteins in Table 27. The amount of each of the Tetz-proteins listed in Table 27 increases when either proteinase K or DNA was added to normal plasma. When proteinase K is added in combination with DNA, the amount of the Tetz-proteins listed in Table 27 decreases relative to when only DNA is added. In plasma cells from patients with cancer, adding proteinase K generally decreases the level of Tetz-proteins listed in Table 27.
sapiens GN = C3
sapiens GN = C3
sapiens
sapiens
sapiens
sapiens GN =
sapiens
sapiens
sapiens
The identification of Tetz-proteins allows for simultaneous evaluation of a large variety of different cancer biomarkers. Such simultaneous analysis can be useful for the diagnostics of neoplastic processes. Analysis of a correlation between Tetz-proteins also known as oncomarkers, and/or other Tetz-proteins, can be used for the development of algorithms for the specific evaluation of certain cancers. Moreover, a correlation between Tetz-protein profile of cancer and non-cancer patients can be studied.
0.5 ml of blood plasma of patient with breast cancer was used and treated with DNA up to the final concentration of nucleic acid up to 1 ng/mL and heated at 100° C. for 2 minutes. Proteins were analyzed with LC/MS analysis, which was conducted using nanoflow UPLC-MS/MS (Thermo Q Exactive HF Orbitrap), in which ultra high-performance liquid chromatography was coupled to tandem mass spectrometry according to the manufacturer's instructions.
Alterations of Tetz-proteins were found following DNA treatment. Moreover, among proteins which amount and coverage were altered, there was a large number of cancer biomarkers that are known to be associated with different cancers.
As a non-limiting example, Complement factor H-related protein is absent in normal patients, is present in cancer plasma and appears in normal plasma after processing with DNA.
Addition of nucleic acids to human fluids and then processing to identify Tetz-proteins allows for evaluating alteration of representation of proteins known an oncomarkers.
0.5 ml of blood plasma of a patient with breast cancer was treated with protease (proteinase K) and heated at 100° C. for 60 minutes. Proteins were analyzed with LC/MS analysis using nanoflow UPLC-MS/MS (Thermo Q Exactive HF Orbitrap) in which ultra high performance liquid chromatography was coupled to tandem mass spectrometry according to the manufacturer's instructions.
Alterations of Tetz-proteins were found following the protease treatment. Moreover, among proteins whose amount and coverage were altered, there was a large number of cancer biomarkers that are known to be associated with different cancers.
Use of proteinases to identify Tetz-proteins allows for evaluating alteration of representation of proteins known an oncomarkers.
Methods
Plasma Samples
Human plasma samples from 5 healthy donors (age: 57-64 years, 40% females) and 5 patients with clinically diagnosed pancreatic ductal adenocarcinoma (age: 56-69 years, 60% females) were obtained from Bioreclamation IVT (NY, USA) and Discovery Life Sciences (Los Osos, Calif.). All patients with pancreatic ductal adenocarcinoma had been diagnosed by histological examination and had not undergone surgical treatment, preoperative chemotherapy or radiotherapy. The basic demographic characteristics of the patients are shown in Table 32. All samples were obtained with prior informed consent at all facilities. Plasma samples were stored at −80° C. until use.
Extracellular DNA
Extracellular DNA was extracted from the matrix of P. aeruginosa ATCC 27853, E. coli ATCC 25922, and Staphylococcus aureus ATCC 29213. All bacterial strains were subcultured from freezer stocks onto Columbia agar plates (Oxoid Ltd., London, England) and incubated at 37° C. for 48 h. To extract the extracellular DNA, bacterial cells were separated from the matrix by centrifugation at 5000 g for 10 min at 4° C. The supernatant was aspirated and filtered through a 0.2-μm-pore-size cellulose acetate filter (Millipore Corporation, USA). eDNA was extracted by using a DNeasy kit (Qiagen), as described by the manufacturer, or by the phenol-chloroform method. Human genomic DNA (Roche Cat #11691112001) was purchased from Sigma (Sigma-Aldrich).
Plasma Exposure to eDNA
DNA was added to plasma samples at the final concentration of 1 μg/mL, incubated at 37° C. for 1 h, and boiled in a water bath at 100° C. for 15 min (by that time all the samples formed clod by coagulated proteins). Samples were cooled at room temperature for 30 min and centrifuged at 5000 g for 10 min at room temperature. The supernatant was aspirated and filtered through a 0.2-μm pore size cellulose acetate filter (Millipore Corporation, USA).
Protein Identification by LS-MS
The filtered protein-containing supernatant was diluted in a final volume of 100 μL using 100 mM ammonium bicarbonate, pH 8, and quantified using a Nanodrop OneC Spectrophotometer (Thermo Fisher Scientific). Cysteine residues were reduced using 5 mM dithiothreitol at room temperature for 1.5 h and alkylated with 10 mM iodoacetamide at room temperature for 45 min in the dark. Proteins were then digested using modified trypsin (Promega, P/N V5113) at a 1:20 (w/w) enzyme:protein ratio for 16 h at 22° C. room temperature. After digestion, peptides were acidified to pH 3 with formic acid and desalted using Pierce Peptide Desalting Spin Columns (P/N 89852), according to the manufacturer's protocol. Eluted, desalted peptides were dried down to completion using a Labconco speedvac concentrator, resuspended in 0.1% formic acid and quantified again using a Nanodrop OneC Spectrophotometer. For sample injection and mass analysis, peptides were diluted to a final concentration of 500 ng/μL using 0.1% formic acid in water to provide a total injection amount of 500 ng in a 1 μL of sample loop. Peptides were separated and their mass analysed using a Dionex UltiMate 3000 RSLCnano ultra-high performance liquid chromatograph (UPLC) coupled to a Thermo Scientific Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer (MS). A 1.5 hr reversed-phase UPLC method was used to separate peptides using a nanoEASE m/z peptide BEH C18 analytical column (Waters, P/N 186008795). The MS method included top 15 data-dependent acquisition for interrogation of peptides by MS/MS using HCD fragmentation. All raw data were searched against the human Uniprot protein database (UP000005640, accessed Apr. 22, 2017) using the Andromeda search algorithm within the MaxQuant suite (v 1.6.0.1). The search results were filtered to a 1% FPR and visualized using Scaffold (v4, Proteome Software).
A cut-off of at least 5 spectral counts per probe was applied for protein selection. The obtained data were used to generate a heatmap. The abundance values were log converted (zero values were replaced with infinitely small number “1”) and plotted with R-statistical computing (www.r-project.org/), using the “levelplot” package. The colour key indicates a range between the lowest (black) and the highest (yellow) values.
Principal components analysis was performed using the prcomp function with default parameters (zero values were replaced with 1) of the R software (www.r-project.org/).
Identification of Prion-Like Domains (PrDs) in Proteins
The presence of prion-like domains in the proteins was assessed using the PLAAC prion prediction algorithm, which establishes the prionogenic nature on the basis of the asparagine (Q) and glutamine (N) content, using the hidden Markov model (HMM). The output probabilities for the PrD states in PLAAC were estimated based on the amino acid frequencies in the PrDs of Saccharomyces cerevisiae. Here, Alpha=0.0 was used, representing species-independent scanning, to identify the PrDs.
Results
eDNA-Induced Alteration of Protein Heat Resistance in the Plasma of Healthy Controls.
The effects of DNA on the thermal behaviour of proteins from the plasma of healthy individuals were first studied. Most proteins were aggregated after boiling, and the supernatant contained heat-resistant fractions of over 100 proteins. Treatment with bacterial and human buffy coat DNA altered the composition of the heat-resistant protein fraction. The levels of which plasma proteins was first verified, identified as heat-resistant before the treatment with DNA, and were increased following DNA exposure in at least one healthy control (Table 28).
The increase in heat-resistant protein fractions following the treatment of plasma with bacterial eDNA was next measured. The highest increase in heat-resistant fractions of different unrelated proteins was registered after the incubation with eDNA of Pseudomonas aeruginosa. Notably, eDNA from different bacteria produced distinct effects. Indeed, the exposure to eDNA from Staphylococcus aureus resulted in a selective increase in heat-resistant APOA2, which was not observed after treatment with eDNA from other bacteria. Under the same conditions, E. coli eDNA increased the heat-resistant fractions of A1AG2, APOB, and C4BP; however, the latter heat-resistant fractions were also increased after exposure to P. aeruginosa eDNA.
Intriguingly, specific proteins that did not exhibit a heat-resistant fraction in untreated plasma samples became heat-resistant following eDNA exposure. Table 29 lists the proteins that displayed such a behaviour in at least one of the plasma samples.
These findings clearly demonstrated that human DNA and eDNA from different bacteria had a distinct influence on the generation of heat-resistant protein fractions. To further analyse the correlation between DNA exposure and acquisition of heat resistance, a heat map was constructed summarizing the impact of different DNAs on the thermal behaviour of proteins (
Plasma exposure to the eDNA of P. aeruginosa resulted in the formation of 12 heat-resistant proteins, while only some of these proteins, namely K1C10, SEPP1, IGLC3, and IF5A1 acquired heat resistance after treatment with the DNA of another gram-negative bacteria. E. coli. The latter, in turn, changed the heat resistance profile of distinct proteins in the same plasma samples. Notably, whereas bacterial eDNA induced heat resistance of a broad spectrum of unrelated proteins, plasma exposure to human DNA only affected the thermal behaviour of a specific group of proteins, i.e., cytoskeletal keratins.
Since prion domains may be responsible for protein heat resistance, the inventors next employed the prion-prediction PLAAC algorithm to verify the presence of PrDs in proteins exhibiting changes in thermal behaviour following DNA treatment.
Only PrDs in CHD7 and K1C10 were found, which became heat-resistant following the exposure to E. coli eDNA and keratins (K2C1, K1C9, K1C10), which acquired heat resistance upon treatment with both P. aeruginosa eDNA and human DNA (Table 30). Notably, these were the only proteins undergoing thermal behaviour alterations following exposure to human DNA.
The association between DNA-induced changes in protein thermal behaviour and human diseases was next analysed. Surprisingly, the majority of these proteins had been found associated with cancer progression and some of them are used as a tumour markers (Table 31).
Intriguingly, some of these cancer-related proteins are also known to be associated with other multifactorial diseases. For example, ITIH4 is associated with schizophrenia and CHD7 is known to be implicated in autism [71-73].
The changes in protein thermal behaviour induced by DNA in normal plasma were then examined and compared the resulting pattern with the heat-resistant proteome of patients with pancreatic cancer (Table 32).
After boiling, the plasma samples of patients with pancreatic cancer were characterized for the presence of heat-resistant proteins. Notably, the majority of these proteins were the same that became heat-resistant in normal plasma exposed to DNA treatment. This might suggest that DNA exposure is responsible for cancer-related alterations in the thermal behaviour of specific proteins.
To further explore the relationship between the heat-resistant proteome of patients with pancreatic cancer and the proteome changes induced by DNA in the plasma of healthy individuals, the scaled spectral counts of the identified heat-resistant proteins of both groups were analysed by principal component analysis (PCA) (
The PCA projection demonstrated that the exposure to bacterial DNA (especially the eDNA of P. aeruginosa), induces, in the proteome of normal plasma, changes in thermal behaviour (
A heat map based on the highest spectral counts relative to heat-resistant proteins confirmed that treatment of normal plasma with eDNA of P. aeruginosa induced a heat-resistant proteome that had a trend (statistically insignificant) more similar to that of plasma from cancer patients than to untreated plasma (
Methods
Plasma Samples
Human plasma samples from 5 healthy donors (age: 57-64 years, 40% females) and 5 patients with clinically diagnosed pancreatic ductal adenocarcinoma (age: 56-69 years, 60% females) were obtained from Bioreclamation IVT (NY, USA) and Discovery Life Sciences (Los Osos, Calif.). All patients with pancreatic ductal adenocarcinoma had been diagnosed by histological examination and had not undergone surgical treatment, preoperative chemotherapy or radiotherapy. The basic demographic characteristics of the patients are shown in Table 4. All samples were obtained with prior informed consent at all facilities. Plasma samples were stored at −80° C. until use.
Nucleases
Proteinase K was purchased from Sigma (Sigma-Aldrich, Cat #P2308).
Plasma Exposure to Proteinase K
Proteinase K was added to plasma samples, incubated at 37° C. for 1 h, and boiled in a water bath at 100° C. for 15 min (by that time all the samples formed clod by coagulated proteins). Samples were cooled at room temperature for 30 min and centrifuged at 5000 g for 10 min at room temperature. The supernatant was aspirated and filtered through a 0.2-μm pore size cellulose acetate filter (Millipore Corporation, USA).
Protein Identification by LS-MS
The filtered protein-containing supernatant was diluted in a final volume of 100 μL using 100 mM ammonium bicarbonate, pH 8, and quantified using a Nanodrop OneC Spectrophotometer (Thermo Fisher Scientific). Cysteine residues were reduced using 5 mM dithiothreitol at room temperature for 1.5 h and alkylated with 10 mM iodoacetamide at room temperature for 45 min in the dark. Proteins were then digested using modified trypsin (Promega, P/N V5113) at a 1:20 (w/w) enzyme:protein ratio for 16 h at 22° C. room temperature. After digestion, peptides were acidified to pH 3 with formic acid and desalted using Pierce Peptide Desalting Spin Columns (P/N 89852), according to the manufacturer's protocol. Eluted, desalted peptides were dried down to completion using a Labconco speedvac concentrator, resuspended in 0.1% formic acid and quantified again using a Nanodrop OneC Spectrophotometer. For sample injection and mass analysis, peptides were diluted to a final concentration of 500 ng/μL using 0.1% formic acid in water to provide a total injection amount of 500 ng in a 1 μL of sample loop. Peptides were separated and their mass analysed using a Dionex UltiMate 3000 RSLCnano ultra-high performance liquid chromatograph (UPLC) coupled to a Thermo Scientific Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer (MS). A 1.5 hr reversed-phase UPLC method was used to separate peptides using a nanoEASE m/z peptide BEH C18 analytical column (Waters, P/N 186008795). The MS method included top 15 data-dependent acquisition for interrogation of peptides by MS/MS using HCD fragmentation. All raw data were searched against the human Uniprot protein database (UP000005640, accessed Apr. 22, 2017) using the Andromeda search algorithm within the MaxQuant suite (v 1.6.0.1). The search results were filtered to a 1% FPR and visualized using Scaffold (v4, Proteome Software).
A cut-off of at least 5 spectral counts per probe was applied for protein selection.
The obtained data were used to generate a heatmap. The abundance values were log converted (zero values were replaced with infinitely small number “1”) and plotted with R-statistical computing (www.r-project.org/), using the “levelplot” package. The colour key indicates a range between the lowest (black) and the highest (yellow) values.
Principal components analysis was performed using the prcomp function with default parameters (zero values were replaced with 1) of the R software (www.r-project.org/).
Results
Proteinase Induced Alteration of Protein Heat Resistance in the Plasma of Healthy Controls
Treatment with microbial proteases changes the composition of the heat-resistant protein fraction, resulting in an increase of certain heat-resistant protein fractions (Table 33).
It was first examined whether viral proteins with prion-like domains can trigger protein in P53-PMCA by monitoring the levels of Thioflavin T (ThT) fluorescence overtime.
The HHV-8 ATCC strain was used. The average kinetics of aggregation of P53 under the treatment with HHV-8, with and without knockout of PrDs contanining proteins, was assayed. The specific proteins knocked out were Human herpes simplex virus 8 RF1 (U5NM22), Human herpes simplex virus 8 LANA (E5L001), and Human herpes simplex virus 8 ORF 73 (A0A0N9S3L8).
A solution of 0.1 mg/ml of monomeric recombinant full-length P53 was subjected to cycles of P53-PMCA either alone (control) or in the presence of 25 μl of various HHV-8 modifications. The experiment was performed at 37° C. in buffer 100 mM PIPES, pH 6.5, 0.5M NaCl. The cycles involved 29 minutes of incubation followed by 1 minute of shaking (500 rpm). The aggregation of the protein was monitored over time by recording thioflavin T (ThT) fluorescence. Each sample was run by duplicate and data shows the average of the two values.
The data is shown in
It was examined whether HIV viral proteins with prion-like domains can trigger Tau protein aggregation in Tau-PMCA by monitoring the levels of Thioflavin T (ThT) fluorescence overtime. A solution of 0.1 mg/ml of monomeric recombinant full-length Tau was subjected to cycles of Tau-PMCA either alone (control) or in the presence of 25 μl of various HIV-1 modifications. The experiment was performed at 37° C. in buffer 100 mM PIPES, pH 6.5, 0.5M NaCl and doing cycles of 29 min incubation and 1 min shaking (500 rpm). The aggregation of the protein was monitored over time by recording thioflavin T (ThT) fluorescence. The HIV-1 ATCC strain was used. Each sample was tested in duplicate.
The data is shown in Table 34, which shows the average kinetics of aggregation of Tau under the treatment with HIV-1 with and without knockout of surface-located proteins containing PrDs. The quantities in Table 34 reflect ThT fluorescence, with the result of each experiment shown. In the “blank” columns, there is no protein aggregation; no tau protein misfolding was observed. In HIV-1 WT, significant misfolding is seen starting at 120 hours, as seen by the increase in ThT fluorescence.
It is clearly seen that wild-type HIV-1 leads to a significant misfolding of Tau protein. Under the same conditions, the mutant HIV-1 strain had much lower proliferative ability, unexpectedly highlighting the role of viral prion-like domains in trans-kingdom misfolding of human proteins.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all values are approximate, and are provided for description.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
This application claims priority to U.S. Provisional Application Ser. No. 62/664,670, filed on Apr. 30, 2018, the contents of which are incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/026272 | 4/8/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62664670 | Apr 2018 | US |
