Patent Application
20220162274
The present invention pertains to protein engineering. In particular, to a method for enhancing the DNA binding affinity of DNA-proximal regions within the globular motif known as “winged” helix-turn-helix (wHTH)—as α1-β1-α2-α3-β2-β3, where the αi are alpha helices and the βj are beta sheets—in the histone H1 protein (also known as linker histone) which in turn is critical for necessary higher-order constraints on chromatin dynamics. In the context of the major wHTH motif, the invention relates in particular to a method for enhancing the DNA-binding affinity of the histone H1 α-helical structural motifs α3 (most preferred, which binds to both nucleosomal and linker DNA), α2 (second most preferred, which binds to nucleosomal DNA), and α1 (third most preferred, which binds to linker DNA, see
The potential of this invention to treat and/or prevent senescence, cancer, and/or age-related health conditions in multicellular species encompasses clinical, cosmetic, industrial, and agricultural applications.
For centuries, intellectual endeavors have entertained the prospect of unlimited lifespan. Today, from services of cryonic suspension and storage of humans and pets to “anti-aging” skin care cosmetics, a variety of industries try to satisfy the increasing human demand to ameliorate or even overcome the accumulation of biological dysfunctions after adulthood that characterizes the senescence process. However, to humans and most other multicellular species, senescence remains an unstoppable process that inexorably leads to the death of the multicellular individual.
On the other hand, the group of diseases called cancer is a major cause of death worldwide, with 9.6 million estimated deaths in 2018. Also, over 70% of deaths from cancer take place in low- and middle-income countries. Additionally, the global economic burden of cancer in 2010 was estimated to be approximately US $1.16 trillion.
Current treatments for cancer include surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy, stem cell transplant, and precision medicine. These treatments mainly aim to prolong the patient's life and in some cases to cure cancer occurrences (especially early detectable cancers). Nevertheless, a comprehensive and effective treatment for cancer, let alone its effective prevention, is still lacking.
Importantly for the present invention, there is a well-known strong positive correlation between cancer incidence and age after adulthood. With respect to said correlation, age-related cancer has been described as the result of a poorly tuned yet strong enough “pushback” of the multicellular individual against its own senescence process. As a consequence, it has been also suggested that stopping senescence and eliminating the incidence of age-related cancer are one and the same technical challenge.
In this context, what is needed is an invention that effectively confers the multicellular individual resistance (being this resistance prophylactic and/or therapeutic) to senescence and/or age-related health conditions, such as age-related cancer.
The specifics of the present invention will be presented in the next section.
With respect to the related state of the art, the patent application WO2005040814 addresses methods and means of cancer detection based on the determination of the presence or amount of post-translational modifications (PTMs) of residues within histone proteins, for example, methylation of lysine residues, in order to assess a cancer condition. Methylated lysine residues, which may be detected in these methods, include, for example, H3 Lys 27, H3 Lys 36, and H4 Lys 20. This document does not address H1 proteins or artificial sequences thereof in terms of their application in the treatment/prevention of senescence or cancer.
Additionally, document WO0151511 addresses the recombinant production of human histone H1 subtypes and their therapeutic uses for cancer, autoimmune diseases, endocrine disorders, and also their use as an antibiotic, where the protein or an active fragment of it is synthesized in E. coli. Importantly, this document never addresses any redesign or artificial sequence of the histone H1.0 or histone H1x protein α-helical motifs and its uses thereof.
WO0172784 shows therapeutic peptides having a motif that binds specifically to non-acetylated H3 and H4 histones for cancer therapy and compositions thereof. This invention is based on the anti-carcinogenic property of a chromatin binding peptide isolated from soybean seed having a highly conserved motif in other chromatin-binding proteins from different species, showing that it can be developed as an in vivo gene silencing technology for biological and medical research. Pharmaceutical compositions useful in retarding, stopping, or reducing various types of cancers are also described. The document never addresses the histone H1.0 or histone H1x proteins or artificial sequences thereof for the treatment/prevention of senescence, cancer, or age-related health conditions.
Finally, U.S. Pat. No. 8,962,562 claims a method for treating thrombocytopenia using at least one human recombinant histone, especially at least one histone H1 subtype. This document focuses on the histone H1.3 protein and in particular does not address the redesign of the histone H1.0 protein or the histone H1x protein nor the redesign of their respective α-helical motifs and uses thereof.
The present invention is directed to a method for producing artificial protein and artificial nucleic acid sequences, where the artificial sequences are useful for the analysis, diagnosis, treatment, and/or prevention of senescence (also known as biological aging) and/or age-related health conditions, such as age-related cancer.
The artificial sequences produced through the method can be used on general research for better understanding of both the senescence and cancer phenomena in multicellular species.
The scope of the invention also encompasses any biomedical, cosmetic, industrial, and agricultural uses of the method according to the present invention.
This invention consists in a method that applies a set of amino acid substitutions, insertions and/or deletions to the wild-type histone H1.0 and histone H1x protein (or any respective protein ortholog) sequences wherein the set of amino acid substitutions, insertions and/or deletions entails an increase in the net electric charge (z) at physiological pH of the artificial-sequence protein DNA-binding regions (in particular, α-helices) with respect to their wild-type counterparts when these artificial-sequence and wild-type protein regions are each in their respective post-translationally unmodified forms or, particularly, when each region is respectively subjected to plausible post-translational modifications (PTMs), effectively creating a “reservoir” of positive electric charge in the artificial-sequence histone H1, which facilitates its electrostatic binding (especially when post-translationally modified in vivo) to the negatively charged DNA.
The present invention also encompasses the artificial sequences produced using the method and the uses thereof.
The method for producing artificial histone H1 sequences is useful for a number of biomedical, cosmetic, industrial, and/or agricultural purposes pertaining to senescence (also known as biological aging) and/or age-related health conditions, such as certain types of cancer.
Arginine is the most basic amino residue, has a high α-helical configuration propensity, and its positive electric charge (i.e., z>0) is virtually permanent at physiological pH.
In its post-translationally unmodified form, the lysine residue is positively charged (i.e., z>0) at physiological pH. Yet, when lysine undergoes post-translational acetylation it suffers a significant decrease in z. If lysine residues are present in histone H1 α-helical motifs then lysine post-translational acetylation is, in principle, possible. Thus, one or more arginine residues substituting a lysine residue known or predicted to undergo post-translational acetylation (or substituting residues proximal to said lysine residue) serve in the method as a “reservoir” of positive electric charge that stabilizes the electrostatic binding affinity of the artificial histone H1.0 and histone H1x proteins to the negatively charged nucleosomal and/or linker DNA.
The asparagine residue has relatively low α-helical configuration propensity yet it is still found in N- and C-terminal regions of α-helical motifs and, in particular, asparagine can be found at the N-terminus of the α3-helix within H1.0 and H1x histones. Asparagine is electrically neutral at physiological pH but it may undergo a deamidation reaction, which converts it to the negatively charged aspartic acid. While infrequent, this deamidation reaction is significantly more likely when the asparagine residue is followed by a glycine residue.
Alanine and methionine are electrically neutral amino acid residues at physiological pH, display a high α-helical configuration propensity and, importantly, are not subject (given their properties and location in the histone H1 globular domain) to PTMs that significantly decrease their net electric charge (z). Thus, under this invention alanine and methionine are adequate substitute residues to be applied in the method to wild-type α-helical motifs.
The proline residue is electrically neutral at physiological pH and hydroxylation is the only PTM proline can be subjected to, which does not decrease its net electric charge (z). However, proline's unique properties make its helix-forming propensity extremely low. Thus, under this invention the proline residue is an adequate substitute only for residues located at the N-terminus of wild-type α-helical motifs (if located at the N-terminus of an α-helical motif, a proline residue is incapable of breaking/kinking said motif and may even stabilize it).
Post-translationally phosphorylated amino acid residues display a decreased electric charge at physiological pH with respect to their respective unmodified form because of the addition of the negatively charged phosphate group. Thus, under this invention, a phosphorylatable amino acid residue (i.e., serine, threonine, tyrosine, and histidine) within an α-helical motif may be deleted or, better yet, substituted with residues such as alanine, methionine, leucine, arginine, or proline (provided proline is substituting a phosphorylatable residue located at the N-terminus of the α-helix). Such an amino acid substitution in the method also creates a “reservoir” of positive electric charge with respect to the substituted wild-type amino acid residue when phosphorylated.
Wild-type α-helical motifs may display aspartic acid or glutamic acid residues, which are negatively charged (i.e., z<0) at physiological pH. Particularly when located nearby DNA-binding amino acid residues, a negatively charged residue can be deleted or, better yet, substituted with a residue such as alanine according to the present invention. A method substituting a negatively charged amino acid residue with one that is not effectively creates a “reservoir” of positive electric charge with respect to the net electric charge (z) of the wild-type α-helical motif at physiological pH.
Importantly, whereas the method entails the application of a set of amino acid substitutions, insertions, and/or deletions applied to wild-type histone H1.0 and histone H1x protein sequences, its preferred embodiment applies only amino acid substitutions (as opposed to amino acid insertions or deletions) at up to eleven specific sites. Alternatively, the method can also apply amino acid substitutions (other than the aforementioned), insertions, and/or deletions to the wild-type histone H1.0 or histone H1x protein sequences as long as the secondary and tertiary protein structures remain biologically functional.
At the multicellular-individual level, this invention is useful for providing significant resistance to senescence (also known as biological aging) and to age-related conditions (e.g., some types of cancer) by stabilizing the electrostatic binding affinity of the histone H1.0 and histone H1x to the negatively charged nucleosomal and/or linker DNA at the chromatin level, which in turn stabilizes the higher-order, histone H1-dependent constraints on chromatin dynamics. Said constraints are critical, when disrupted or dissipated, for the senescence and the age-related cancer phenomena.
It is also important to emphasize that, whereas the artificial histone H1.0 and histone H1x proteins produced by the method can be natively synthesized by genetically modified non-human species, for humans it is principally envisioned the extrinsic delivery of synthetic mRNA (encoding the artificial-sequence histone H1.0 and histone H1x proteins) to the cells. The future development of an effective “histone H1.0/H1x replacement/supplement therapy” (via gene suppression techniques and/or in vivo mRNA delivery) is plausible because (i) histone H1 proteins display a very high turnover rate in chromatin (the mean residence time of a histone H1 protein at its binding site has been estimated to be approximately 3 minutes) and (ii) both H1.0 and H1x histone variants are not tissue-specific, which should facilitate the delivery of the required synthetic mRNA in vivo.
For a fuller understanding of this invention, reference is made to the following description and accompanying drawing, in which:
In particular, the present invention provides a method for producing artificial protein and artificial nucleic acid sequences (such as RNA or DNA) for two histone H1 variants or any of their respective orthologs, wherein the method entails the application of a set of amino acid (aa) substitutions, insertions, and/or deletions to the respective wild-type (wt) protein sequence at clearly defined sites, and wherein the method entails the satisfaction of a very specific electrochemical condition for each artificial protein sequence produced by the method with respect to its wild-type counterpart.
In a preferred embodiment of the invention, the method entails the application of a set of amino acid substitutions, insertions, and/or deletions which in turn entails an increase in the net electric charge (z) (at physiological pH) of the globular domain in the artificial-sequence histone H1 with respect to its wild-type counterpart when both globular domains are each in their respective post-translationally unmodified forms or when each globular domain is subjected to plausible post-translational modifications (PTMs), in order to stabilize the electrostatic binding affinity of the artificial-sequence histone H1 (in particular when subjected to PTMs in vivo) to the negatively charged DNA. This effect is possible by virtue of the net electric charge (z) as a function of (i) the side chain and (ii) the ability to undergo PTMs for each amino acid residue placed by the method in the artificial protein sequence and/or those for the amino acid residues displaced by the method from the wild-type protein sequence.
In a more preferred embodiment of the invention, the method entails the application of a set of amino acid substitutions, insertions, and/or deletions to the α3, α2, and/or α1-helix subsequences within the histone H1.0 (also known as histone H1°; H1(0); H5; H1δ; RI H1; or H1 histone family, member 0) protein sequence, to the α3, α2, and/or α1-helix subsequences within the histone H1x (also known as histone H1.10 or H1 histone family, member X) protein sequence, and to the α3, α2, and/or α1-helix subsequences within any respective histone H1.10/H1x ortholog sequence—because said α3, α2, and α1 subsequences correspond to the structural motifs that bind to nucleosomal and/or linker DNA in chromatin.
In an even more preferred embodiment of the invention, the method entails the application of a set of at least one and up to eleven amino acid substitutions at eleven respective specific sites spanning the α3 amino acid subsequence, the N-terminal region of the α2 subsequence, and the N-terminal region of the α1 subsequence, where all three subsequences are within the full sequence of the histone H1.0, histone H1x, and of any respective orthologous protein. This embodiment is even more preferred because amino acid substitutions (as opposed to insertions or deletions) are less likely to disrupt the α-helical geometry and the eleven amino acid substitution sites are those most proximal (within the α-helical motifs) to nucleosomal or linker DNA. The selection of the set of at least one and up to eleven amino acid substitutions includes at least one amino acid substitution in the α3-helix subsequence. In a further embodiment of the invention, the method comprises at least two more amino acid substitutions, where at least one of the remaining substitutions is made to a α2, and/or a α1-helix subsequence within the histone H1.
Therefore, the invention relates in particular to the redesign of the histone H1 α-helical motifs α3 (most preferred, which binds to both nucleosomal and linker DNA), α2 (second most preferred, which binds to nucleosomal DNA), and α1 (third most preferred, which binds to linker DNA), through amino acid sequence modification (by amino acid substitutions, insertions, and/or deletions).
The method for producing artificial histone H1 protein sequences, according to the present invention, is useful for treating and/or preventing senescence, cancer, and/or age-related health conditions.
In this invention, reference is made to the following naturally occurring α-amino acids (aa): alanine (IUPAC one-letter symbol: A, three-letter symbol: Ala), cysteine (C, Cys), aspartic acid (D, Asp), glutamic acid (E, Glu), phenylalanine (F, Phe), glycine (G, Gly), histidine (H, His), isoleucine (I, Ile), lysine (K, Lys), leucine (L, Leu), methionine (M, Met), asparagine (N, Asn), proline (P, Pro), glutamine (Q, Gln), arginine (R, Arg), serine (S, Ser), threonine (T, Thr), valine (V, Val), tryptophan (W, Trp), and tyrosine (Y, Tyr).
Reference is made to amino acid substitutions with the nomenclature XW#XA, where # is the site (identified by counting from the translation initiator Met residue numbered as +1) occupied by a wild-type (wt) amino acid residue XW to be substituted by an amino acid residue XA according to the present invention.
Reference is made to the net electric charge (IUPAC symbol: z) defined as the algebraic sum of the charges present at the surface of a molecule divided by the elementary charge of the proton. In this context, reference is also made in this application to zP, hereby defined as the net electric charge of a molecule at physiological pH (i.e., when pH-7.4). Consequently, a higher zP implies a more positive (or, equivalently, less negative) net electric charge in a molecule at physiological pH. It has to be noted that the application of the set set of amino acid substitutions, insertions and/or deletions according to the present invention entails an increase in the net electric charge of the molecule at physiological pH (zP), it may also modify its net electric charge (z) at other other pH values or ranges.
Reference is made to nucleosomal DNA (also known as core nucleosomal DNA), understood as the DNA that is left-hand wrapped around the histone octamer forming a complex known as the nucleosome core particle (NCP), which is the building block of chromatin.
Reference is made to linker DNA, understood as the DNA that extends in between nucleosome core particles. Importantly for this invention, the phosphate group repeated across the backbone of nucleic acids in particular makes both nucleosomal and linker DNA negatively charged at physiological pH.
Reference is made to the histone H1 (also known as linker histone) protein, which constitutes one of the five major histone protein families necessary for the formation of chromatin in the eukaryotic cell. Specific regions within the histone H1 protein bind to nucleosomal and/or linker DNA, which in turn stabilizes the higher-order constraints on chromatin dynamics.
The histone H1 family comprises a number of variants. These histone H1 variants are encoded by paralog gene families and classified under a phylogeny-based nomenclature. However, they are also grouped according to protein biosynthesis in terms of its relationship with the cell cycle and its tissue specificity.
Reference is made to a major structural motif known as “winged” helix-turn-helix (wHTH) in the form of α1-β1-α2-α3-β2-β3 where the αi motifs are alpha helices and the βj motifs are beta sheets, which characterizes the globular domain of the histone H1 protein. Importantly for this invention, the histone-H1 α1, α2, and α3 helices are motifs with amino acid residues known to be proximal (and thus more likely to bind) to nucleosomal and/or linker DNA.
Reference is also made to post-translational modifications (PTMs), which are covalent and typically (but not necessarily) enzymatic modifications undergone by amino acid residues in proteins following protein biosynthesis.
Histone H1 proteins are subjected to PTMs. Some of the most common PTMs undergone by histone H1 proteins are acetylations and phosphorylations. Lys acetylation is known to lower the otherwise positive electric charge of the Lys residue at physiological pH, in turn decreasing the Lys-mediated electrostatic binding affinity of histone H1 to the negatively charged nucleosomal and linker DNA. The post-translational phosphorylation of amino acid residues such as Ser, Thr, Tyr, and His is also known to decrease the electrostatic DNA binding affinity of the histone H1.
When proximal to DNA-binding regions, the negatively charged Asp and Glu residues may reduce, by electrostatic repulsion, the binding affinity of those regions to nucleosomal and linker DNA.
In general, any wild-type amino acid residue (even a residue of null z, sometimes used itself as a substitute residue in other instances) located in a DNA-binding region of the histone H1 protein may be substituted in the method according to this invention with an amino acid residue such that zP is increased in that region, thereby stabilizing the binding affinity to the negatively charged nucleosomal and linker DNA when that region and/or others in the histone H1 protein are subjected to PTMs in vivo.
When the present invention applies a set of at least one and up to eleven amino acid substitutions to a wild-type histone H1.0 or histone H1x protein sequence, adequate substitute residues include but are not limited to Arg, Ala, Met, Asn, and Pro (the latter being adequate only for substituting an amino acid residue located at the N-terminal site of an α-helical motif).
Since the present invention aims to elicit a significant phenotypic change in the entire multicellular individual in its adult form, the method must target a histone H1 variant that (i) accumulates in terminally differentiated cells and (ii) is synthesized in the whole body of the individual. In other words, the targeted histone H1 variant must be both replication-independent (i.e., synthesized throughout the cell cycle) and somatic.
Importantly, only two histone H1 variants embody both characteristics: the H1.0 (also known as histone H1°; H1(0); H5; H1δ; RI H1; or H1 histone family, member 0) variant, which is common to all multicellular species and the H1x (also known as histone H1.10 or H1 histone family, member X) variant, which is unique to vertebrate species.
Within the histone H1.0/H1x structure, the α1 helix is 13 amino acid residues long, the α2 helix is 12 amino acid residues long, and the α3 helix is 16 amino acid residues long.
The histone H1.0/H1x α1 motif (in particular its N-terminal region) binds mainly to linker DNA, the α2 motif (in particular its N-terminal region) binds mainly to nucleosomal DNA and, importantly, the α3 binds to both nucleosomal and linker DNA (see
Amino acid residues in the histone H1.0/H1x α1, α2, and/or α3 motifs can be post-translationally modified. In particular, specific residues can be acetylated or phosphorylated, which entails a decrease in zP for the α-helical motifs, which in turn decreases the electrostatic binding affinity of the histone H1.0/H1x protein to both nucleosomal and/or linker DNA.
The invention presented here corrects for the PTM-dependent decrease in electrostatic DNA binding affinity and/or for an intrinsically low DNA binding affinity—the latter caused mainly by negatively charged amino acid residues—in the wild-type protein without impairing the function of the α-helical motifs within the histone H1.0/H1x protein nor the function of the protein as a whole.
Specifically, this invention claims a method for producing artificial protein sequences and artificial nucleic acid sequences for the histone H1.0 and histone H1x variants and their orthologs, which are useful for the analysis, diagnosis, treatment, and/or prevention of senescence (also known as biological aging) and/or age-related health conditions, such as some types of cancer. This invention also encompasses the artificial protein sequences and artificial nucleic acid sequences produced by the method of the invention.
In the method the application of a set of amino acid substitutions, insertions, and/or deletions to produce artificial histone H1.0 and histone H1x protein sequences (from the three respective wild-type, α-helical subsequences) entails an increase of zP in the artificial-sequence α3, α2, and/or α1 motifs with respect to their wild-type counterparts when the artificial and wild-type motifs are each in their respective post-translationally unmodified forms or when each is subjected to plausible PTMs.
The application of a set of amino acid substitutions only—as opposed to insertions or deletions, because substitutions are more likely to preserve the secondary structure and overall function of the wild-type protein in the artificial-sequence protein derived from it while creating in the artificial-sequence protein a new function for the multicellular individual—applied to a wild-type histone H1.0 or histone H1x informs the most preferred embodiment of this invention.
When the artificial-sequence α3, α2, and/or α1 motifs undergo post-translational modifications in vivo, the set of amino acid substitutions, insertions, and/or deletions applied by the method to produce the artificial-sequence α3, α2, and/or α1 motifs effectively creates a “reservoir” of positive electric charge that stabilizes or enhances the electrostatic binding affinity of the artificial-sequence histone H1.0/H1x protein to the negatively charged nucleosomal and/or linker DNA.
This stabilization or enhancement of the electrostatic binding affinity of the artificial-sequence histone H1.0/H1x protein to nucleosomal and/or linker DNA in turn stabilizes the higher-order constraints on chromatin dynamics in the terminally differentiated cells of the multicellular individual, which in turn translates into significant resistance to senescence and/or to age-related health conditions for the multicellular individual.
For a person skilled in the art, it would be evident that, although the intracellular activity is given by the artificial histone H1 protein, these proteins can be obtained through (i) artificial DNA sequences prepared using any technique available in the state of the art such as genome editing or plasmid systems and (ii) artificial RNA sequences, where all these artificial nucleic acid sequences encoding the artificial protein sequences produced by the claimed method, as well as their complementary reverse in the case of the DNA sequences, are considered within the scope of the present invention.
The non-triviality and high specificity of the set of amino acid substitutions, insertions, and/or deletions—in terms of location (DNA-binding protein regions) and required effect (increase of z in DNA-binding protein regions)—that must in turn be applied to the wild-type sequence of highly specific, functionally well-defined proteins (histone H1.0 and/or histone H1x) constitutes a clear inventive step in this patent application.
The method according to this invention is intended to induce resistance and/or protection against senescence (also known as biological aging) and/or age-related health conditions in multicellular species by producing artificial protein sequences and nucleic acid sequences for the histone H1 variants H1.0 (also known as histone H1°; H1(0); H5; H1δ; RI H1; or H1 histone family, member 0) and H1x (also known as histone H1.10 or H1 histone family, member X), and comprises the steps of:
The present invention encompasses artificial histone H1.0 or H1x protein sequences for inducing resistance and/or protection against senescence, and/or age-related health conditions—which include but not are not limited to age-related cancer, atherosclerosis and cardiovascular disease, arthritis, cataracts, osteoporosis, type-2 diabetes, hypertension, Alzheimer's disease, benign prostate hyperplasia, hearing disability, age-related macular degeneration, neurodegenerative diseases, degenerative diseases, immune senescence diseases, skin aging, and skin wrinkles—where the artificial protein sequence contains a set of at least one amino acid substitutions, insertions, and/or deletions to the DNA-binding site of the histone H1.0 or H1x proteins in the α-helical regions, where the substitutions, insertions, and/or deletions do not alter the structure of the α-helical motifs and also entail an increase in the net electric charge (z) of the resulting artificial-sequence protein. The net electric charge (z) is estimated particularly at physiological pH.
The DNA binding sites are located in the first, second, and/or third (counting from N- to C-terminus) α-helices of the histone H1.0 and histone H1x proteins.
The amino acid sequence that corresponds to the first α-helix, denoted by α1, of the wild-type histone H1 protein counterpart is identical or homologous to SEQ. ID No. 1 if the wild-type histone variant is H1.0 or to the SEQ. ID No. 4 if the wild-type histone variant is H1x.
The amino acid sequence that corresponds to the second α-helix, denoted by α2, of the wild-type histone H1 protein counterpart is identical or homologous to SEQ. ID No. 2 if the wild-type histone variant is H1.0 or to SEQ. ID No. 5 if the wild-type histone variant is H1x.
The amino acid sequence that corresponds to the third α-helix, denoted by α2, of the wild-type histone H1 protein counterpart is identical or homologous to SEQ. ID No. 3 if the wild-type histone variant is H1.0 or to SEQ. ID No. 6 if the wild-type histone variant is H1x.
In an embodiment of the invention, the set of amino acid modifications corresponds to at least one to up to eleven amino acid substitutions within the binding site in the α-helical motif.
In a further embodiment of the invention, the eleven amino acid substitution sites α1 to α11 comprise at least one substitution for each of the first, second, and third α-helical motifs selected from: (S1,α3,12), (S2,α3,13), (S3,α2,1), (S4,α1,1), (S5,α3,1), (S6,α2,3), (S7,α3,3), (S8,α3,5), (S9,α3,9), (S10,α2,2) and (S11,α3,11); where each triplet shows the substitution site, the α-helical motif and the relative position (counting from N- to C-terminus); where the substitute amino acid residue are selected from alanine, methionine, leucine and arginine, for any substitution site or proline for substitution sites α3 and/or α4.
For a proper zP comparison between artificial and wild-type protein sequences, the same dissociation-constant data—i.e., the same set of pKa values for the α-carboxylic acid group, α-ammonium group, and side chain group (if applicable) of each amino acid residue—must be used as calculation base.
The artificial-sequence histone H1.0 and histone H1x proteins according to the present invention, when synthesized by engineered cells (e.g., via genome editing or synthetic mRNA delivery) or administered extrinsically to cells (if extracellular histone H1 cytotoxicity can be countered) so that in treated multicellular individuals the artificial-sequence proteins reach abundance levels comparable to those of their wild-type protein counterparts in untreated individuals, confer the treated individuals significant resistance to senescence and/or age-related health conditions.
The artificial histone H1.0 and histone H1x protein sequences, the synthetic or recombinant nucleic acid sequences encoding said artificial protein sequences, and the methods for producing such sequences according to this invention are useful for analyzing and/or diagnosing senescence and/or age-related health conditions in multicellular species.
The artificial histone H1.0 and histone H1x protein sequences according to this invention are useful for inducing resistance and/or protection against senescence and age-related health conditions in multicellular species. Said resistance and/or protection includes, but is not limited to, the analysis, diagnosis, treatment and/or prevention of senescence and/or other age-related health conditions, such as certain types of cancer.
The artificial histone H1.0 and histone H1x protein sequences—and the synthetic or recombinant nucleic acid sequences encoding them—according to this invention can be used on biomedical, cosmetic, industrial, and agricultural applications.
The method of the invention was tested in vivo on a simple organism, such as C. elegans, in order to verify its effectiveness, and as can be seen in detail in examples 1 and 11, a C. elegans organism was obtained featuring only three amino acid substitutions in the sequence of its histone H1.X protein (ortholog of the human histone H1.0) and displating great resistance to senescence, which translates to a very significant increase in the survival rate. So much so that by day 14 only 50% of the wild type survived and 100% of the mutants (hil-1 gene), and by day 24 there were no wild-type left alive, while the worms which synthesized the mutant histone H1.X protein developed in accordance with the method of the present invention, showing a survival rate above 98%. Based on the results, where 98% of the mutants (subjected to only 3 amino acid substitutions in their histone H1.X protein) are still alive, whereas 100% of the wild-type individuals are dead.
For a person skilled in the art, it would be evident that, although the intracellular activity is given by the artificial histone H1 protein, these proteins can be obtained through (i) artificial DNA sequences prepared using any technique available in the state of the art such as genome editing or plasmid systems and (ii) artificial RNA sequences, where all these artificial nucleic acid sequences encoding the artificial protein sequences produced by the claimed method, as well as their complementary reverse in the case of the DNA sequences, are considered within the scope of the present invention.
The following examples are intended to illustrate the present invention and they cannot be used for limiting its scope.
Example 1: Application of the most preferred embodiment of the method for producing an artificial protein sequence for the somatic, replication-independent histone H1 variant in the model organism Caenorhabditis elegans.
C. elegans H1.X
H. sapiens H1.0
C. elegans H1.X
C. elegans
Examples 2-3: Application of the most preferred embodiment of the method for producing two artificial sequences for the mouse (Mus musculus) histone H1.0 protein.
M.musculus H1.0
H. sapiens H1.0
M. musculus H1.0
Examples 4-5: Application of the most preferred embodiment of the method for producing one artificial sequence for the human histone H1.0 protein and one artificial sequence for the human histone H1x protein.
H.sapiens H1.0
H. sapiens Hlx
Examples 6-10: Since a protein ortholog of the human histone H1.0 can be found in all multicellular species and a protein ortholog of the human histone H1x can be found in all vertebrate species, the method informing the present invention can produce artificial histone H1.0 sequences for any multicellular species and artificial histone H1x sequences for any vertebrate species. Five examples of artificial histone H1.0/H1x protein sequences produced with the method claimed in this invention, using its most preferred steps, for different species are shown in TABLE 1.
Rattus norvegicus
Rattus norvegicus
Nothobranchius furzeri
Drosophila melanogaster
Arabidopsis thaliana
Example 11: In vivo testing of the method for conferring C. elegans resistance to senescence.
For a person skilled in the art it would be obvious that, given the well-known signs of senescence in C. elegans observable shortly after the individual reaches its adult form, the increased lifespan in the hil-1 mutant strain with respect to the wild-type C. elegans (strain N2) also implies the hil-1 mutant strain is significantly resistant to senescence, thereby demonstrating the industrial applicability of the present invention in vivo.
This application claims the benefit of Provisional U.S. Patent Application No. 62/803,987, filed on Feb. 11, 2019. This provisional application is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/051098 | 2/11/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62803987 | Feb 2019 | US |
