Patent Application
20160340656
Peptides derived from GSE24.2 have applications in the biotechnology and pharmaceutical sectors for treating diseases and/or manifestations caused by and/or related to oxidative stress and/or damage to DNA such as inflammation or cellular ageing.
GSE24.2 is a peptide corresponding to the protein dyskerin, which is part of the telomerase complex. GSE24.2 is able to reactivate telomerase in pathological and physiological situations; in diseases with telomerase defects GSE24.2 activity increases the viability of cells derived from patients.
International patent application: WO2007/090911 A1 “Sequence of nucleotides and peptides GSE24.2 of dyskerin, which can induce telomerase activity, Method for obtaining same, Therapeutic compositions and Applications thereof” reveals the use of GSE24.2 for reactivating telomerase activity either in a medicine or a pharmaceutical composition. This international patent application identifies a sequence of 55 amino acids as the original functional unit. This invention describes new peptides of smaller size derived from the original sequence as well as new uses and applications thereof such as protection against oxidative stress and damage to DNA, both processes intimately related to inflammatory and cellular ageing processes.
Furthermore, this invention provides variants of these peptides derived from GSE24.2 that include nuclear localisation sequences enabling higher and better access to the nucleus, which is where they must exercise their biological activity, increasing their efficacy and reducing the need for encapsulation of the original sequence of 55 amino acids.
This invention provides a compound that reduces or decreases production of free radicals and/or damage to the structure of cellular DNA, where this compound is based on a fragment of the nucleotide or amino acid sequence of peptide GSE24.2 of dyskerin, capable of restoring normal cellular redox balance and/or correcting damage to cellular DNA. Thus, the invention addresses the problem of providing new therapeutic tools for the treatment of diseases involving an increase in cellular oxidative stress and/or damage to the structure of DNA such as arteriosclerosis, Parkinson's disease, myalgic encephalopathy, Alzheimer's disease, ataxia telangiectasia, congenital dyskeratosis and ageing.
This patent describes internal sequences of peptide GSE24.2 (SEQ ID No. 1) that have higher biological activity than the complete sequence and its already known fragments; furthermore, to improve their activity, nuclear transport sequences have been added that additionally improve the activity of the peptides by improving their internalisation by cells and increasing their presence in the cell nucleus.
Peptide GSE24.2 (with sequence SEQ ID No. 1) and derived peptides have biological activities not previously described such as the reduction of the free radical content by cells and therefore have activities of reducing oxidative stress and reducing structural damage to DNA caused by mutations in its recognition machinery; all this increases viability of cells exposed to various agents causing the effects previously mentioned. This activity has been tested in some pathologies such as ataxia telangiectasia and congenital dyskeratosis; but this also enables its application in neurodegenerative diseases, muscular degeneration (Duchenne dystrophy), progeroid syndromes, hypersensitivity to light, genetic instability caused by mutations or external agents; all applications not previously described for this peptide.
The basis of this invention is that the inventors have tested the ability of peptides derived from GSE24.2 for reducing the level of free radicals and the damage to DNA present in various human diseases or derived from exposure to agents such as radiation or genotoxic agents. Therefore, peptides derived from GSE24.2 of this invention as well as GSE24.2 itself can be applied to all these diseases caused or involving an accumulation of damage to DNA or an increase in free radicals such as: diseases of accelerated ageing such as Werner syndrome or Hutchison-Gilford syndrome; diseases where there is damage to DNA caused by mutations in the recognition and processing machinery such as ataxia telangiectasia, xeroderma pigmentosum, Rothmund-Thomson syndrome, Nijmegen breakage syndrome; neurodegenerative diseases such as for example, Huntington's, Alzheimer's, Parkinson's diseases; diseases involving muscular degeneration such as Duchenne muscular dystrophy; cardiovascular or lung diseases such as pulmonary fibrosis or arteriosclerosis; also diseases involving a major inflammatory component in their pathology such as rheumatoid arthritis. Applications of these peptides also include those related to tissue engineering and cell culture and other biotechnological applications. These peptides may also have applications in the cosmetics industry such as, for example, in the treatment of damage caused by ageing in the skin.
The inventors have produced small sized peptides derived from GSE24.2 (SEQ ID No. 1) with the ability to enter the cell and that have higher biological activity than the original peptide, identifying them in bacterial lysates of bacteria that express the full sequence of GSE24.2. These lysates were analysed by HPLC prior to the identification of their sequences. Five (5) peptides in addition to GSE24.2 were identified in lysates and their sequences corresponded to SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6.
Table 1 shows a listing of the sequences and correspondences with the peptides described in this invention.
The biological activity of the peptides identified in the bacterial lysates was studied in a DNA damage repair test. Of these, peptide GSE4 (SEQ ID No. 2), which is included in the TRUBL domain, substantially improved the activity of the full domain (SEQ ID No. 4, GSE2) and of GSE24.2 (SEQ ID No. 1); this activity was still further improved by the insertion of a sequence for nuclear localisation in the carboxyl terminal position (but not in the N-terminal position). Given the small size of the peptides identified in this invention, it is not necessary to use cell capture systems such as liposomes to introduce them into the cell; this is a substantial improvement compared to that used for the full peptide GSE24.2 (SEQ ID No. 1) or for the TRUBL domain (GSE2, SEQ ID No. 4) where liposomes are required. These new peptides, therefore, provide a more reproducible and stable system for therapeutic purposes than the current. Furthermore, these peptides can be obtained by chemical synthesis, simplifying the process of obtaining them. These peptides have functions analogous to those of GSE24.2 and therefore it is possible to use them in equivalent applications.
This invention identifies the minimum unit or minimum polypeptide with activity that is included in the GSE24.2 peptide of sequence SEQ ID No. 1 and that consists of a fragment of 11 amino acids of SEQ ID No. 1 and that comprises at least SEQ ID No. 2. This peptide constitutes the minimum unit responsible for GSE24.2 activity of reducing free radical production and/or damage to the structure of DNA in a cell.
Thus, a first aspect of the invention refers to a polypeptide comprising at least a fragment of the sequence of the peptide GSE24.2 with SEQ ID No. 1 able to reduce the production of free radicals and/or the damage to cellular DNA structure and where this fragment comprises, at least, SEQ ID No. 2, except when this polypeptide consists of the peptide GSE24.2 (SEQ ID No. 1) or a fragment of it with sequence SEQ ID No. 4 (GSE2).
In a preferred embodiment, the polypeptide has an amino acid sequence that comprises a fragment of the peptide GSE24.2 selected from at least one of the group formed by: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 6.
When at least one of the polypeptides previously defined are introduced or its expression is induced into the interior of the cell, preferably into the interior of a eukaryotic cell such as a human cell, it is able to cause a reduction in the production of free radicals and/or damage to the DNA of this cell. In this way, on quantifying the levels of free radicals in a live cell by any of the known methods such as for example by the compound dihydroethidium (DHE, Hydroethidine), the production of free radicals in the cell can fall depending on the sequence of this polypeptide until reaching physiological levels with respect to a cell of the same type to which it has not been introduced or in which its expression has not been induced. Similarly, when the damage to cellular DNA is quantified by any of the existing methods such as, for example, the study of the hybridisation signal of cells with an antibody against phosphorylated histone H2AX in position Ser139 (γ-H2AX), this damage to DNA can be observed to reduce by 50% depending on the sequence of the polypeptide with respect to a cell of the same type to which it has not been introduced or in which its expression has not been induced.
Taking into account that the action of the polypeptides defined in this aspect of the invention can take place in the cell nucleus, in order to facilitate their expression or localisation in the nucleus and/or improve the activity of the polypeptides, this polypeptide, apart from the fragment of the GSE24.2 peptide with activity, can also comprise at least a nuclear localisation sequence (NLS) linked to at least one of the carboxyl and amino terminal ends of this fragment, and more preferably to the carboxyl terminal end.
In the scope of this invention, the nuclear localisation sequence is considered to be a group of amino acids capable of directing the localisation of the peptide to be bound to the cell nucleus; this sequence can be either a sequence for transport to the nucleus or a sequence for transport to the nucleolus. Preferably, the nuclear localisation sequence is a nuclear localisation sequence that is found in gene DKC1 from which the sequence of GSE24.2 is derived such as, for example, the nuclear transport sequence KRKR (NLS1) or the nucleolar transport sequence KKEKKKSK (NLS2), and more preferably it is the nuclear transport sequence KRKR (NLS1).
Thus, in a preferred embodiment of the polypeptide of this aspect of the invention, when it comprises a nuclear localisation sequence linked to the active fragment of GSE24.2, this polypeptide consists of an amino acid sequence selected from the group composed of: SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14. In a more preferred embodiment, this polypeptide consists of an amino acid sequence selected from the group composed of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 13 and SEQ ID No. 14. In a yet more preferred embodiment, the polypeptide consists of a sequence that is selected from the group composed of SEQ ID No. 7 and SEQ ID No. 8.
Throughout this document, any of the polypeptides defined in this first aspect of the invention can be equally referred to as “polypeptide of the invention” or “peptide of the invention”.
The polypeptides of the invention can be obtained by the normal procedures known in the art for peptide synthesis: by insertion into expression vectors or by chemical synthesis. Chemical synthesis procedures of peptides comprise both synthesis in solution or liquid phase and solid phase peptide synthesis (SPPS). Preferably, solid phase peptide synthesis would be used, using any of the methods known in the state of the art such as Fmoc and t-Boc.
Another aspect of the invention refers to a polynucleotide that comprises a sequence that codes for at least one of the polypeptides of the invention such as any of those previously defined. This polynucleotide, also referred to in this document as polynucleotide of the invention, enables the expression of a polypeptide that is able to reduce production of free radicals and/or damage to the structure of DNA in a eukaryotic cell, preferably a human cell, and can refer to a polynucleotide of DNA, cDNA or RNA. Preferably, this polynucleotide comprises a sequence that codes for at least one active fragment of the sequence of peptide GSE24.2 with SEQ ID No. 1 and where this fragment comprises at least SEQ ID No. 2, except when this polynucleotide codes for peptide GSE24.2 (SEQ ID No. 1) or a fragment of it with SEQ ID No. 4 (GSE2).
In a preferred embodiment, the polynucleotide of the invention comprises a sequence that codes for a fragment of peptide GSE24.2 selected from at least one of the group formed by: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 6.
Taking into account that the action of the polypeptide that is coded by the polynucleotide of the invention can take place in the cell nucleus, similarly as indicated for the polypeptide of the invention, the polynucleotide sequence of the invention can also comprises at least one nuclear localisation sequence (NLS) bound to at least one of the 3′ or 5′ ends of the sequence of the fragment of peptide GSE24.2, and more preferably bound to the 3′ end.
Preferably, the nuclear localisation sequence chosen for the polypeptide of the invention is a nuclear localisation sequence directed at the nucleus or at the nucleolus, and more preferably, it is in the gene DKC1 from which the sequence of GSE24.2 is derived. Preferred examples of nuclear transport sequences of gene DKC1 are the nuclear transport sequences that code for the peptide sequence KRKR (NLS1) or the nucleolar transport sequences that code for the peptide sequence KKEKKKSK (NLS2), and preferably it is a nuclear transport sequence that codes for the peptide sequence KRKR (NLS1).
In a preferred embodiment, when the polynucleotide of the invention comprises a nuclear localisation sequence linked to the sequence coding for the active fragment of GSE24.2, this polynucleotide consists of a sequence that codes for a polypeptide selected from the group composed of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14. In a more preferred embodiment, it consists of a sequence that codes for a polypeptide selected from the group composed of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 13 and SEQ ID No. 14. In a yet more preferred embodiment, it consists of a sequence that codes for a polypeptide selected from the group composed of SEQ ID No. 7 and SEQ ID No. 8.
The polynucleotide of the invention as any of those described above can be obtained by an expert through the use of techniques widely known in the state of the art (Sambrook et al. “Molecular cloning, a Laboratory Manual” 2nd ed., Cold Spring Harbor Laboratory Press, N.Y., 1989 vol 1-3). The sequences of these polynucleotides can be integrated in a gene expression vector that enables regulation of expression in suitable conditions inside the cells.
Therefore, another aspect of the invention refers to an expression vector that comprises a polynucleotide of the invention or a polynucleotide sequence that codes for a polypeptide of the invention and that enables the expression of a polypeptide that is able to reduce the production of free radicals and/or damage to the structure of DNA in a eukaryotic cell, preferably a human cell, hereafter the vector of the invention.
In general, according to this invention, the expression vector further comprises, in addition to the polynucleotide described in the invention, a promoter that directs its transcription (for example, pT7, plac, ptrc, ptac, pBAD, ret, etc.), to which it is operationally linked, and other necessary or appropriate sequences that control and regulate this transcription and, if applicable, the translation of the product of interest, for example, transcription initiation and termination signals (tlt2, etc.), polyadenylation signal, replication origin, ribosome binding sequences (RBS), sequences encoding transcription regulators (enhancers), transcription silencers (silencers), repressors, etc. Examples of suitable expression vectors can be selected in accordance with the conditions and needs of each specific case from expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. that may additionally contain labels used for selecting cells transfected or transformed with the gene or genes of interest. The choice of vector will depend on the host cell and the type of use to be made of it. Therefore, according to a particular embodiment of this invention, this vector may be a plasmid or viral vector. This vector may be obtained by conventional methods known to those skilled in the subject in the same way that for the transformation of micro-organisms and eukaryotic cells various widely known methods may be used: chemical transformation, electroporation, microinjection, etc., described in various manuals (Sambrook et al. “Molecular cloning, a Laboratory Manual” 2nd ed., Cold Spring Harbor Laboratory Press, N.Y., 1989 vol 1-3).
The polypeptide of the invention, the polynucleotide of the invention and/or the gene expression vector defined in this invention can be used as a medicine in a treatment and/or prevention procedure for a human being affected by a disease caused by an increase in oxidative stress and/or damage to cellular DNA, preferably through its biological action in cells affected by an increase in free radical levels and/or damage to the structure of their DNA, preferably human cells; it may also be used as a biotechnological tool in cell culture systems or in vitro tissue engineering to improve cell viability. However, this invention also considers that the complete sequence of peptide GSE24.2 (SEQ ID No. 1) and/or its 18-amino acid fragment SEQ ID No. 4 (GSE2) can be used for this purpose. It is considered that biotechnological and pharmaceutical tools as well as therapy procedures described in this invention are sufficiently known by an expert in the art so that such tools can be developed without excessive effort.
Thus another aspect of the invention refers to the use of a polypeptide, or of a polynucleotide or vector that codes for this polypeptide, where this polypeptide comprises at least SEQ ID No. 2 (GSE4) and that is a fragment of peptide GSE24.2 (SEQ ID No. 1) and is able to reduce the production of free radicals and/or damage to the structure of cellular DNA, in a medicine preparation or pharmaceutical composition, for the treatment of, at least, one disease caused by an alteration, preferably an increase, of oxidative stress and/or damage to cellular DNA. By way of an illustration and without limiting the scope of the invention, this disease may belong to the following group: congenital dyskeratosis, a neurodegenerative disease (such as Alzheimer's, Parkinson's, Huntington's, cerebellar ataxia or spinal medulla degeneration), Cri du Chat syndrome, ataxia telangiectasia, Nijmegen breakage syndrome, Bloom syndrome, Werner syndrome, Fanconi anaemia, ulcerative colitis, vascular ageing, arteriosclerosis, preferably atherosclerosis, cancer, Duchenne muscular dystrophy, progeroid syndromes, light hypersensitivity, genetic instability caused by mutation or external agent, Hutchison-Gilford syndrome, xeroderma pigmentosum, Rothmund-Thomson syndrome, pulmonary fibrosis and diseases with chronic inflammation such as, for example, rheumatoid arthritis.
In a preferred embodiment of this aspect of the invention, the disease is at least selected from the group composed of: congenital dyskeratosis, a neurodegenerative disease (including for example Alzheimer's, Parkinson's, cerebellar ataxia or spinal medulla degeneration), Cri du Chat syndrome, ataxia telangiectasia, Nijmegen breakage syndrome, Bloom syndrome, Werner syndrome, Fanconi anaemia, ulcerative colitis, vascular ageing, arteriosclerosis, preferably atherosclerosis and cancer.
According to an aspect of the invention, the medicine or pharmaceutical composition comprises a polypeptide and/or polynucleotide or expression vector that codes for it, where this polypeptide comprises a sequence selected from, at least, one of the group composed of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5 and/or SEQ ID No. 6. However, yet more preferable embodiments are those where the polypeptide consists of a sequence equal to or less than 25 amino acids preferably selected from at least one of the group composed of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5 y SEQ ID No. 6.
In another aspect of the invention, the medicine or pharmaceutical composition comprises a polypeptide and/or a polynucleotide or expression vector that codes for it, where the polypeptide additionally comprises at least one nuclear localisation sequence, preferably bound at the carboxyl terminal end, preferably selected from at least one of the group composed of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14. Yet more preferably, the polypeptide sequence is chosen from the group composed of SEQ ID No. 7 and SEQ ID No. 8.
Another object of the invention is a pharmaceutical composition or medicine, preferably for the treatment of diseases, disorders or pathologies caused by a change in the content of free radicals inside the cell, causing oxidative stress associated with and/or caused by damage to the structure of cellular DNA, hereafter the pharmaceutical composition of the invention, that comprises at least one polypeptide, polynucleotide and/or expression vector able to reduce the production of free radicals and/or damage to the structure of cellular DNA, in a therapeutically effective amount together with, optionally, one or more adjuvants and/or pharmaceutically acceptable vehicles, and that is able to regulate and maintain the redox balance and correct damage to DNA. In the scope of this invention, this pharmaceutical composition or medicine can equally be referred to as the “pharmaceutical composition or medicine of the invention”.
The pharmaceutically acceptable adjuvants and vehicles that can be used in these compositions are adjuvants and vehicles known by experts in the field and commonly used in the preparation of therapeutic compositions. By way of example, these may be nanoparticles or liposomes, which help absorption and internalisation of molecules.
In the meaning used in this description, the expression therapeutically effective amount refers to the amount of polypeptide of the invention, or of a molecule or compound that enables the expression of a nucleotide sequences that codes for this polypeptide, able to reduce production of free radicals and/or damage to the structure of cellular DNA, calculated to cause the desired effect and that, in general, would be determined, among other factors, by the properties of the compounds, including age, patient status, severity of the alteration or disorder, and the route and frequency of administration.
In a particular embodiment, this therapeutic composition is prepared in the form of a solid or aqueous suspension in a pharmaceutically acceptable diluent. The therapeutic composition provided by this invention can be administered by any appropriate administrative route, so the composition would be formulated in a suitable pharmaceutically acceptable form for the chosen administration route. In a particular embodiment, administration of the therapeutic composition provided by this invention is by the parenteral route, oral route, intraperitoneal route or subcutaneous route. A review of various pharmaceutical forms of administration of medicines and of the excipients necessary for obtaining them can be found, for example, in Tratado de Farmacia Galénica, C. Faulí i Trillo, 1993, Luzán 5, S. A. Ediciones, Madrid.
In a preferred embodiment, the pharmaceutical composition or medicine comprises a polypeptide of the invention that is selected from at least one of the group composed of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14; or a polynucleotide or vector that codes for this polypeptide. More preferably, the polypeptide that comprises or is coded for is selected from at least one of the group composed of: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 13 and SEQ ID No. 14; and more preferably from at least one of the group composed of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8.
In another preferred embodiment, the pharmaceutical composition or medicine comprises a polypeptide of the invention that contains an active fragment of peptide GSE24.2 that is selected from at least one of the group composed of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 6; or a polynucleotide or vector that codes for this polypeptide.
In another preferred embodiment, the pharmaceutical composition or medicine comprises a polypeptide of the invention that contains an active fragment of peptide GSE24.2 bound to a nuclear localisation sequence, preferably bound to the carboxyl terminal end and is selected from at least one of the group composed of SEQ ID No. 7 and SEQ ID No. 8; or a polynucleotide or vector that codes for this polypeptide.
The pharmaceutical composition of this invention can be used in a treatment method either alone or together with other pharmaceutical compounds.
Another aspect of the invention is therefore the use of the pharmaceutical composition of the invention, hereafter the use of the pharmaceutical composition of the invention, in a method of treatment or prevention in an individual, preferably a human being, affected by a disease, disorder or pathology caused by an alteration in the content of free radicals inside the cell, causing oxidative stress and/or caused by damage to the structure of DNA.
Preferably, the disease or pathology to be treated is one of those defined in the uses of the composition mentioned above.
Another aspect of the invention refers to the use of a polypeptide that comprises a fragment of the sequence of peptide GSE24.2 (SEQ ID No. 1) with SEQ ID No. 2 able to reduce production of free radicals and/or damage to structure of cellular DNA, or of the polynucleotide or vector that codes for it, in tissue engineering and cell culture to improve cell viability. Preferably, this polypeptide consists of at least one selected from the group composed of: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14.
Throughout the description and the claims, the word “comprises” and its variants does not exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and characteristics of the invention will emerge, partly from the description and partly from the practice of the invention. The following examples and figures are provided for illustration purposes only and are not to be considered as limiting the present invention.
To perform this test, cell lines from patients with ataxia telangiectasia (AT, from the Coriell repository) with a mutation in the gene ATM and denominated AT3487 were used. Cell line C-1787 was used as the control cell line. The cells were infected with the bicistronic lentiviral vector pCMV-GFP-GSE24.2. After 48 hours of infection the cells were fixed with formaldehyde and then permeabilized by treating them with Triton X-100. The damage to DNA was estimated by studying the signal after hybridisation of the cells with an antibody against histone H2AX Ser139 and subsequent hybridisation with an antibody bound to fluorescein. The number of foci with stain for histone H2AX/nucleus was determined by confocal microscopy in a total of 200 cells and the data represented as a percentage of cells with a specific number of foci.
Next, a similar experiment was carried out but determining the damage to DNA by hybridisation with an antibody that recognises protein pCHK2 phosphorylated on Thr68, and the data indicated that in this case 25% fewer AT cells showed basal damage after expressing GSE24.2 and high levels of damage to DNA were reduced by 13% (
One of the characteristics of AT cells is an increase in free radical content. This is due to the failure of function of protein ATM, which is able to maintain the redox balance in normal cells when the cells are exposed to environmental stress such as in vitro culture. In order to verify if expression of GSE24.2 has some impact on the regulation of free radical levels, we used the compound dihydroethidium (Hydroethidine), which enables quantifying the levels of free radicals in living cells. For this experiment, we used control lymphoblasts AT736C and a line of AT lymphoblasts AT3189-P. The latter were infected with a lentiviral vector expressing GSE24.2, pCMV-GFP-GSE24.2, or an empty vector expressing only GFP (GFP). Three weeks after infection, the cells were loaded with the DHE reagent for 30 minutes and the proportion of cells with different free radical content was determined by flow cytometry. The results showed (
One of the characteristics of AT patient cells is a higher activation of p38 MAP kinase due to the failure of function of the ATM protein. This increase in activation of p38 triggers the process of cell death in AT cells and therefore a reduction in the number of neuronal cells. Therefore we checked if the expression of GSE24.2 was able to reduce the activation of p38 in cell lines from AT patients. AT719 cells were infected with a lentiviral vector expressing GSE24.2, pCMV-GFP-GSE24.2, or an empty vector expressing only GFP (GFP) and protein extracts were obtained from both cells lines three weeks after infection. The normal lymphoblasts cell line AT736 was used as negative control. The extracts were resolved on polyacrylamide gels and the proteins transferred to nitrocellulose filters for subsequent hybridisation with antibodies. An antibody that detects phosphorylated p38 protein was used and later a rehybridisation was performed with an antibody against non-phosphorylated p38 protein as a loading control. The signal obtained in both hybridisations was quantified with the NIH-Image program and relative values between the phosphorylated and non-phosphorylated proteins obtained for the control cell line were considered as 1.
Mutations in gene ATM sensitise cells of AT patients to treatment with ionizing radiation or radiomimetic drugs such as bleomycin. In order to verify if the protective effect induced by GSE24.2 in cells of AT patients also extends to radiomimetic agents, AT719 patient cells expressing pCMV-GFP-GSE24.2 or an empty vector expressing only GFP (GFP) were treated with increasing concentrations of bleomycin for 72 hours. After this time, cell viability was evaluated using MTS reagent. AT736C cells were used as a control. The results showed that expression of GSE24.2 was able to increase viability of AT719 cells to levels similar to control cells, whereas AT719 or AT719GFP cells were more sensitive than controls (
Bacterial lysates were obtained from RosettaGami2 bacteria expressing the bacterial vector pGATEVGSE24-2. To do this, the bacteria were sonicated in saline medium with protease inhibitors and the fusion protein expressed by the bacterial plasmid was purified on glutathione sepharose columns. The lysates were subjected to HPLC to separate the peptides from the other cellular proteins using columns for separation by size and charge. The resulting fractions were sequenced using a sequencer that uses a method derived from the Edman method and in commercial use. The various peptides found in bacterial lysates of bacteria expressing GSE24.2 were obtained by chemical synthesis and tested for the activity of repairing damage to DNA using F9A353V cells carrying the most frequent mutation (A353V) in patients with congenital dyskeratosis. To do this, various peptides were lipofected using Proteojuice reagent in F9A353V cells and F9 control cells (cells without damage) were used for control of damage. An unrelated peptide was used as a negative control and the full GSE24.2 as a positive control. After quantifying the Western blots (
In order to optimise the operating conditions of peptide GSE4, different amounts of peptide GSE4 were transfected into F9A353V cells and the amount of damage to DNA was determined using a specific antibody as was performed in
The other way of quantifying the activity of peptide GSE4 was to evaluate its ability to increase telomerase activity in VA13 cells. To do this, these cells were transfected with 8 μg of the two peptides GSE4 and GSE5. After 48 hours, the cells were processed to test the telomerase activity by the TRAP method.
As the peptide denominated GSE4 was smaller than the peptides with ability to repair telomerase activity, we studied if it had the same activity as peptide GSE24.2 for attenuating oxidative stress, expression of proinflammatory cytokines and repairing the senescence in ataxia telangiectasia cells.
As the peptide denominated GSE4 was the smallest sized of the peptides with ability to inhibit damage to DNA, we studied if another peptide with a similar sequence localised in the sequence of peptide GSE24.2 had the same activity or if that activity was specific to GSE4. Peptide SGSE4 (SEQ ID No. 15) is similar to GSE4 in sequence (amino acids LDPK/LDKP that constitute consensus sequences for pseudouridine synthases) and size and is found in the TRUBII domain of dyskerin. Cell lines AT2078 of patients with ataxia telangiectasia were used for this test. Cell line C-1787 was used as the control cell line. The cells were infected with the bicistronic lentiviral vector pCMV-GFP-GSE4, SGSE4 or GFP. After 48 hours of infection the cells were fixed with formaldehyde and then permeabilized by treating them with Triton X-100. The damage to DNA was estimated by studying the signal after hybridisation of the cells with an antibody against histone H2AX Ser139 and subsequent hybridisation with an antibody bound to fluorescein. The number of foci with stain for histone H2AX per nucleus was determined by confocal microscopy in a total of 200 cells and the data represented as a percentage of cells with a specific number of foci.
An important aspect to be considered for peptide GSE24.2 is its subcellular localisation after it is expressed. Therefore various constructions were obtained, placing two nuclear localisation signals NLS1 and NLS2 in the 5′ and 3′ region of GSE24.2. NLS1 is a signal present in the sequence of gene DKC1 that carries the protein to the nucleus and sequence NLS2 mediates transport to the nucleolus.
The expression plasmid in eukaryotic cells, pCDNA3myc, was used, which contains a tag of the gene c-myc in position 5′ merged with the sequence GSE24.2. Various constructions were obtained that are shown in
With the aim of evaluating biological activity of the constructions containing the signals NLS1-3 and N LS2-3, they were transfected into 293T cells together with the reporter gene of the promoter of c-myc and the luciferase activity activated by both constructions GSE24.2NLS1-3 and GSE24.2NLS2-3 was evaluated in relation to GSE24.2.
Given the ability of sequence NLS1 to contribute to the activity of GSE24.2, a synthetic peptide into which this sequence was placed in the 3′ region of peptide GSE4 was constructed as this was the peptide for which the greatest telomerase activation was obtained. The peptides obtained by chemical synthesis were labelled with fluorescein to facilitate their visualisation in a fluorescence microscope. Two experiments were performed in parallel, in one the peptides were transfected using Proteojuice reagent and in the other they were added directly to the culture medium. The results showed that both GSE4 and GSE4NLS1 (SEQ ID No.7) entered the cells by using Proteojuice reagent (
| Number | Date | Country | Kind |
|---|---|---|---|
| P201331573 | Oct 2013 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2014/070803 | 10/24/2014 | WO | 00 |
