WO2021009299A1

WO2021009299A1 - Bcl-xl:fkbp12 fusion proteins suitable for screening agents capable of slowing down the aging process

Info

Publication number: WO2021009299A1
Application number: PCT/EP2020/070174
Authority: WO
Inventors: Chloé JAMES; Muriel PRIAULT
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université De Bordeaux; Centre National De La Recherche Scientifique (Cnrs); Chu De Bordeaux
Priority date: 2019-07-17
Filing date: 2020-07-16
Publication date: 2021-01-21

Abstract

The molecular damages leading to the deterioration of cellular and tissue functions occur at different rate in different people. Thus methods for screening anti-aging agents are highly desirable. The present fulfils this need by providing fusions proteins wherein the Bcl-xL protein is fused to a stability-affecting protein. By inducing the degradation of the fusion protein and then restoring said expression is thus possible to rejuvenate a marker of cell aging. The apparition of the mono-deamidated form will indeed correlate with the aging progression of the cells and thus will offer a reliable system of identifying agents that are capable of slowing down the aging process. The preferred fusion protein is a fusion of Bcl-XL with the mutant L106P of FKBP12.

Description

BCL-XLFKBP12 FUSION PROTEINS SUITABLE FOR SCREENING AGENTS CAPABLE OF

SLOWING

DOWN THE AGING PROCESS

FIELD OF THE INVENTION:

The present invention is in the field of medicine.

BACKGROUND OF THE INVENTION:

The molecular damages leading to the deterioration of cellular and tissue functions occur at different rate in different people. There is currently high societal expectation to decipher the mechanisms of aging and open the possibility of anti-aging medicine. Deamidation is a post-translational modification (PTM) that converts asparagine residues into aspartate/isoaspartate and glutamine residues into glutamate. Although deamidases have been characterized in bacteria or viruses, in eukaryotic cells deamidation is thought to proceed through a spontaneous, non-enzymatically catalyzed slow chemical process (for review, see

Unlike most PTMs, deamidation is an irreversible process that permanently edits the genetic information initially translated into proteins. Therefore, pending a destruction by proteolysis balanced by an active neosynthesis, deamidated proteins are bound to build up with age, and cells are bereft of means to mitigate the modifications entailed. From the functional point of view, deamidation is essentially referred to as a detrimental process because of the structural consequences entailed by Iso-Asp occurrence (the protein backbone is rerouted to incorporate a supplementary carbon atom), and because of the loss of catalytic activity ^2_4, protein cleavage ⁵ or even degradation ⁶ documented (see ⁷ for review). Therefore, the time-dependent accumulation and functional decay imparted by deamidation led to the“molecular clock” concept, whereby deamidation regulates cellular functions through the control of either the amount or the activity of a protein, or both. Sequence analyses in higher eukaryotes predict that hundreds of proteins are eligible for deamidation, with a fair number experimentally verified. Despite the indisputable fact that deamidation wields potent functional alterations to so many proteins, this PTM remains largely overlooked because its occurrence is not easily detectable. Each Asn or Gin deamidation only confers a mass increase of 1 Da, and an additional negative charge.

BC1-XL is an oncogene that was characterized in 1993 as a member of the Bcl-2 family (for B-cell lymphoma 2) (Boise et ah, 1993). Within the family, Bcl-xL shows the unique trait to be eligible for deamidation. While single deamidation of Asn52 was shown to improve the autophagic response in cells confronted to nutrient starvation (Beaumatin et al., 2016), the double deamidation of Bcl-xL on Asn52 and Asn66 was essentially shown to occur in response to DNA damage, and to cripple its anti-apoptotic activity. It was suggested that deamidated forms of Bcl-xL could be used as a biomarker of cell ageing (Beaumatin, Florian, et al. "Bcl- xL deamidation and cancer: charting the fame trajectories of legitimate child and hidden siblings." Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1864.10 (2017): 1734-1745).

SUMMARY OF THE INVENTION:

As defined by the claims, the present invention relates to fusion proteins and uses thereof for screening agents capable of slowing down the aging process.

DETAILED DESCRIPTION OF THE INVENTION:

The molecular damages leading to the deterioration of cellular and tissue functions occur at different rate in different people. Thus methods for screening anti-aging agents are highly desirable. The present fulfils this need by providing fusions proteins wherein the Bcl-xL protein is fused to a stability-affecting protein. By inducing the degradation of the fusion protein and then restoring said expression, it is thus possible to rejuvenate a marker of cell aging. The apparition of the mono-deamidated form will indeed correlate with the aging progression of the cell and thus will offer a reliable system of identifying agents that are capable of slowing down the aging process.

The first object of the present invention relates to a fusion protein wherein the Bcl-xL protein is fused to a stability-affecting protein.

As used herein, the terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

The term“fusion polypeptide” or“fusion protein” means a protein created by joining two or more polypeptide sequences together. The fusion polypeptides encompassed in this invention include translation products of a chimeric gene construct that joins the nucleic acid sequences encoding a first polypeptide with the nucleic acid sequence encoding a second polypeptide. In other words, a“fusion polypeptide” or“fusion protein” is a recombinant protein of two or more proteins which are joined by a peptide bond or via several peptides. The fusion protein may also comprise a peptide linker between the two domains. As used herein, the term“linker” refers to a sequence of at least one amino acid that links the first polypeptide to the second polypeptide in a fusion protein.

As used herein, the term“Bcl-xL” has its general meaning in the art and refers to the human protein also known as B-cell lymphoma-extra large, having a sequence as set forth in SEQ ID NO: 1 and homologs and orthologs thereof.

SEQ ID NOl >sp I Q07817 I B2CL1_HUMAN Bcl-2-like protein 1 OS=Homo sapiens OX=9606 GN=BCL2L1 PE=1 SV=1 Asn52 and Asn66 are shown in bold and underlined.

MSQSNRELWDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAV GAT GHSSSLDAREVI PMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQWNELFRDGVNWG RIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQ ERFNRWFLTGMTVAGWLLGSLFSRK

As used herein, the term“stability-affecting protein” refers to a ligand-binding domain that was reselected to confer either stability or instability to the entire fusion protein, depending on the presence or absence of a stabilizing ligand. A feature of the conditional protein stability system is that the stability-affecting protein is of a“single ligand-single domain” type, which minimizes the number of components in the system.

In some embodiments, the stability-affecting protein is a variant of the FKBP12 protein. As used herein, the term“FKBP12” or“FKBP” has its general meaning in the art and refers to a 12 kDa protein that binds to the small-molecules rapamycin and FK506. An exemplary amino acid sequence is represented by SEQ ID NO:2. According the present invention the positions of the amino acids in the FKBP 12 protein are numbered as follows: the methionine residue at position 1 in SEQ ID NO:2 corresponds to the position 0 according to numbering system of the present invention. Accordingly, the leucine at position 106 corresponds to the leucine residue at position 105 in SEQ ID NO:2.

SEQ ID NO: 2 >sp | P62942 | FKB1A HUMAN Peptidyl-prolyl cis-trans

isomerase FKBP1A OS=Homo sapiens OX=9606 GN=FKBP1A PE=1 SV=2

MGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLTI SPDYAYGATGHPGI I PPHATLVFDVELLKLE

As used herein, a“variant” is a protein having an amino acid sequence that does not occur in nature, as exemplified by sequences in GenBank.

As used herein, the term“FKBP12 variant” refers to a protein wherein one or more amino acid residues, e.g., at positions 15, 24, 25, 36, 60, 100, and 106, are substituted for an amino acid other than the amino acid in the FKBP12 protein. More particularly, the stability- affecting protein is the mutant L106P of FKBP12 (12 kD), which is rapidly degraded in mammalian cells. The FKBP12 protein itself is well-characterized for its ability to form a tight complex with immunosuppressive drugs, e.g., FK505 and rapamycin (Pollock and Clackson 2002). This L106P mutant is a very strong potent destabilizing domain. When cells expressed a yellow fluorescent protein (YFP) fusion of this mutant, 1-2% of normal YFP levels was observed in the absence of its stabilizing ligand. The addition of the stabilizing ligand stabilized the L106P-YFP fusion in a dose-dependent fashion, and full stability was achieved with 1 mM of the ligand (Banaszynski et al. 2006).

In some embodiments, the stability-affecting protein has an amino acid sequence as set forth in SEQ ID NO:3.

SEQ ID NO: 3 > L106P variant of FKBP12

MGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV

GQRAKLTI SPDYAYGATGHPGI I PPHATLVFDVELLKPE

According to the invention, the fusion protein of the present invention is produced by conventional synthesis methods that typically involve recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. Recombinant DNA technology may be indeed employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides. A variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell systems infected with virus expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al, 2000. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art and a briefly described herein below. Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxyl ation, glycosylation, phosphorylation, lipidation and acylation. Post- translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

Thus a further object of the invention relates to an isolated, synthetic or recombinant nucleic acid molecule encoding for a fusion protein of the present invention.

As used herein, a“nucleic acid molecule” or“polynucleotide” refers to a DNA molecule (for example, but not limited to, a cDNA or genomic DNA). The nucleic acid molecule can be single-stranded or double-stranded.

The term“isolated” when referring to nucleic acid molecules or polypeptides means that the nucleic acid molecule or the polypeptide is substantially free from at least one other component with which it is associated or found together in nature.

In some embodiments, the nucleic acid of the present invention is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

So, another object of the invention relates to a vector comprising a nucleic acid of the invention.

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. The vectors may further comprise one or several origins of replication and/or selectable markers. The promoter region may be homologous or heterologous with respect to the coding sequence, and provide for ubiquitous, constitutive, regulated and/or tissue specific expression, in any appropriate host cell, including for in vivo use. Examples of promoters include bacterial promoters (T7, pTAC, Trp promoter, etc.), viral promoters (LTR, TK, CMV- IE, etc.), mammalian gene promoters (albumin, PGK, etc), and the like. Examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication- defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.

Another object of the present invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid molecule and/or a vector according to the invention.

As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

Examples of host cells include eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). The construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques. The fusion protein of the present invention is expected to work in various eukaryotic cells, including those of humans, primates, rodents, dogs, cats, horses, cows, sheep, insects, amphibians, and apicomplexan parasites. The cells may be in culture or in a living organism. In some embodiments, the expression of the natural Bcl-xL protein is optionally repressed in the host cell. In some embodiments, the expression of Bcl-xL is repressed by using an endo nuclease. In some embodiments, the expression of Bcl-xL is repressed by using a CRISPR-associated endonuclease. CRISPR/Cas systems for gene editing in eukaryotic cells typically involve (1) a guide RNA molecule (gRNA) comprising a targeting sequence (which is capable of hybridizing to the genomic DNA target sequence), and sequence which is capable of binding to a Cas, e.g., Cas9 enzyme, and (2) a Cas, e.g., Cas9, protein. The targeting sequence and the sequence which is capable of binding to a Cas, e.g., Cas9 enzyme, may be disposed on the same or different molecules. If disposed on different molecules, each includes a hybridization domain which allows the molecules to associate, e.g., through hybridization. Artificial CRISPR/Cas systems can be generated which inhibit Bcl-xL, using technology known in the art, e.g., that are described in U.S. Publication No. 20140068797, WO2015/048577, and Cong (2013) Science 339: 819-823. Other artificial CRISPR/Cas systems that are known in the art may also be generated which inhibit Bcl-xL, e.g., that described in Tsai (2014) Nature Biotechnol, 32:6 569-576, U.S. Pat. Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359, the contents of which are hereby incorporated by reference in their entirety. Such systems can be generated which inhibit Bcl-xL, by, for example, engineering a CRISPR/Cas system to include a gRNA molecule comprising a targeting sequence that hybridizes to a sequence of the Bcl-xL gene. In some embodiments, the gRNA comprises a targeting sequence which is fully complementarity to 15-25 nucleotides, e.g., 20 nucleotides, of the Bcl-xL gene. In some embodiments, the 15-25 nucleotides, e.g., 20 nucleotides, of the Bcl-xL gene, are disposed immediately 5' to a protospacer adjacent motif (PAM) sequence recognized by the Cas protein of the CRISPR/Cas system (e.g., where the system comprises a S. pyogenes Cas9 protein, the PAM sequence comprises NGG, where N can be any of A, T, G or C).

According to the present invention, the host cell of the present invention offers a system wherein the expression of the Bcl-xL protein is finely controlled. For instance, in the absence of a stabilizing ligand, the destabilizing domain mediates the degradation of the entire fusion protein. On the contrary, the addition of an appropriate ligand stabilizes the destabilizing domain, greatly reducing degradation of the fusion protein. Thus in some embodiments, the stabilizing ligand may be contacted with the host cell to stabilize the fusion protein until the cell reaches a particular stage of development, at which time withdrawal of the ligand results in a the rapid degradation of the fusion protein. As used herein,“degradation” or“destruction” of a protein means its hydrolysis into smaller proteins or amino acids, such as by the cellular proteasome.

According to the present invention the stabilizing ligand is a small molecule ligand. As used herein, the term“small molecule ligand” is a discrete small-molecule, well known in the pharmaceutical and material sciences, which is to be distinguished from, e.g., a polypeptide or nucleic acids, which is a polymer consisting of monomeric subunits. Small molecule ligands may be naturally-occurring or synthetic as exemplified by pharmaceutical products, laboratory reagents, and the like.

In some embodiments, the stabilizing ligand of the present invention is Shieldl . As used herein, the term“shieldl” refers to a synthetic small molecule that binds to a FKBP variant. Yhe IUPAC name of shieldl is l-[2-(3,4,5-trimethoxy-phenyl)-butyryl]-piperazine-2- carboxylic acid 3-(3,4-dimethoxy-phenyl)-l-[3-(2-morpholin-4-yl-ethoxy)-phenyl]-propyl ester).

In some embodiments, the present nucleic acid molecule of the present invention also allows the creation of non-human transgenic animals harboring engineered alleles that direct the expression of the fusion protein of the present invention. Expression of the fusion protein may be driven by an endogenous promoter (e.g. the Bcl-xL promoter), for designing a spatial and temporal expression pattern in the organism, e.g. similarly as for the Bcl-xL protein. The stabilizing ligand may be administered regularly from an early age (including in utero) to stabilize the fusion protein until the mice achieve a specified age, at which time withdrawal of the ligand results in a the rapid degradation of the fusion protein. This method is reversible, simply by reinitiating the administration of the ligand, allowing the rapid, reversible, and conditional control of protein function in a complex system.

The host cells and non-human transgenic animals of the present invention are particularly suitable for screening anti-aging agents and/or rejuvenating agent.

Thus a further object of the present invention relates to a method for identifying a substance useful as anti-aging agent comprising the steps of i) providing a host cell or a non human transgenic animal of the present invention, ii) contacting said host cell or non-human transgenic animal with an amount of the stabilizing ligand for a sufficient time for allowing the expression of the fusion protein of the present invention, iii) withdrawing the stabilizing ligand for a sufficient time for allowing the degradation of the fusion protein of the present invention, iv) contacting the host cell or non-human transgenic animal of the present invention with an amount of a test substance in the presence of an amount of the stabilizing ligand for a sufficient time for allowing the expression of the fusion protein of the present invention v) performing a time-course analysis to monitor the rate of mono-deamidation of fusion protein in which the ability of the test substance to act as an anti-aging agent is indicated by the slowdown detection of the mono-deamidated form as compared to the deamidation rate observed in the absence of the test substance and (vi) selecting the substance that slowdown the apparition of the mono- deamidated form.

A further object of the present invention relates to a method for identifying a substance useful as rejuvenating agent comprising the steps of i) providing a host cell or a non-human transgenic animal of the present invention, ii) contacting said host cell or non-human transgenic animal with an amount of the stabilizing ligand for a sufficient time for allowing the expression of the fusion protein of the present invention and reaching a pre-treatment steady state of deamidation, iii) withdrawing the stabilizing ligand for a sufficient time for allowing the degradation of the fusion protein of the present invention, iv) contacting the host cell or non human transgenic animal of the present invention with an amount of a test substance in the presence of an amount of the stabilizing ligand for a sufficient time to allow the expression of the fusion protein of the present invention and to reach a post-treatment steady state v) comparing the steady state of deamidation of the fusion protein before and after exposure to the test substance; whereby the rejuvenating effect of the substance is indicated by the decreased accumulation of the mono-deamidated form after treatment as compared to the deamidation steady state observed before treatment and finally (vi) selecting the substance that resets the epigenetic clock of the cell, ie rejuvenate cellular parameters enough to impair the accumulation of the mono-deamidated form.

As used herein, the term“mono-deamidated form” refers to the fusion protein wherein the asparagine residue at position 52 in the Bcl-xL protein is substituted by an aspartic acid residue or an isoaspartic acid residue as a result of cellular spontaneous deamidation reaction.

According to the invention, the detection of mono deamidated form is determined by any routine technique well known in the art. Typically, the detection is performed as described in the EXAMPLE. In some embodiments, the detection of the mono-deamidated form is determined by using an antibody that is specific for said form. In some embodiments, the antibody is labelled with a tag to facilitate the detection of the mono-deamidated form. As used herein, the terms "label" or "tag" refer to a composition capable of producing a detectable signal indicative of the presence of the mono-deamidated form. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In some embodiments, a flow cytometric method is involved. As used herein, the term "flow cytometric method" refers to a technique for counting cells of interest, by suspending them in a stream of fluid and passing them through an electronic detection apparatus. Flow cytometric methods allow simultaneous multiparametric analysis of the physical and/or chemical parameters of up to thousands of events per second, such as fluorescent parameters. Modern flow cytometric instruments usually have multiple lasers and fluorescence detectors. A common variation of flow cytometric techniques is to physically sort particles based on their properties, so as to purify or detect populations of interest, using "fluorescence-activated cell sorting". As used herein, "fluorescence-activated cell sorting" (FACS) refers to a flow cytometric method for sorting a heterogeneous mixture of cells from a biological sample into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell and provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. For example, fluorescence activated cell sorting (FACS) may be therefore used and typically involves use of a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores (such as a BD Biosciences FACSCanto™ flow cytometer). The cytometric systems may include a cytometric sample fluidic subsystem, as described below. In addition, the cytometric systems include a cytometer fluidically coupled to the cytometric sample fluidic subsystem. Systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and/or speakers, softwares (e.g. (Flowjo, Laluza.... ), data input devices, e.g., interface ports, a mouse, a keyboard, etc., fluid handling components, power sources, etc.

The test substance of the invention may be selected from a library of substances previously synthesised, or a library of substances for which the structure is determined in a database, or from a library of substances that have been synthesised de novo. The test substance may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) organic or chemical substances.

According to the present invention, the agents identified by the screening methods of the present invention are particularly suitable for increasing mitochondrial biogenesis and function, reducing ROS levels, extending life span of senescent cells and post-mitotic cells such as neuron cells. In some embodiments, the agent identified by the screening method of the present invention may find various applications. In some embodiments, the agents identified by the screening methods of the present invention may be particularly suitable for preventing age- related disorders. As used herein, the term“age-related disorder” or“age-related disease” refers to disorders or diseases in which aging is a major risk factor. Based on the type of diseases, age- related diseases or disorders include three main types: (1) abnormal poliferative diseases, such as cancer; (2) degenerative diseases, including neuron degenerating disease (Alzheimer's, Parkinson's, stroke), myocardial infarction, heart failure, atherosclerosis, hypertension, osteoarthritis, osteoporosis, sarcopenia, loss of bone marrow, rheumatoid arthritis, degraded immune function, diabetes, idiopathic pulmonary fibrosis, age-related macular degeneration; and (3) function decreasing disorders, including declines in testosterone, estrogen, growth hormone, IGF-I, reduced energy production and so on. Based on the type of cells involved, age- related diseases or disorders can also be classified as two main classes: (1) in postmitotic cells: neuron degeneration (Alzheimer's, Parkinson's, stroke), sarcopenia (loss of muscle), cardiovascular diseases (heart failure, myocardial infarction); and (2) in mitotic cells: loss of bone marrow, degraded immune function, diabetes, idiopathic pulmonary fibrosis, age-related macular degeneration, rheumatoid arthritis, osteoarthritis, osteoporosis, atherosclerosis, and hypertension. More specifically, Age-related diseases or disorders associated with mitochondrial dysfunction or/and telomere dysfunction include, but are not limited to, cancer, osteoarthritis, age-related macular degeneration, idiopathic pulmonary fibrosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, skin aging, cataract, multiple sclerosis, Sjogren, Rheumatoid arthritis, atherosclerosis, myocardial infarction, heart failure, hypertension, stroke, diabetes mellitus, osteoporosis, and obesity. In some embodiments, the agents identified by the screening methods of the present invention may also find interest in cosmetics. For instance the agent may be suitable for preventing age-related skin and hair damages. More particularly the agent may be applied to the skin, scalp or hair, and may be used in order to prevent skin aging, and improve wrinkles and rough skin.

As used the term“anti-aging agent” or“rejuvenating agent” refers to agent used against “age-related disorder” or “age-related disease”. Examples of “anti-aging agent” or “rejuvenating agent” include but are not limited to rapamycin, metformin, fisetin, acarbose, 17- a-estradiol, spermidine, resveratrol.

The methods and compositions described herein may be packaged together with instructions for use, as in a kit of parts. Preferred kits of parts include an amount of fusion proteins and/or nucleic acid molecules and/or host cells of the present invention, and instructions for use. In some embodiments, the kit of the present invention comprises an amount of stabilizing ligands (e.g. shieldl). The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Mice tissues were collected from animals at the indicated ages (in weeks) and ground to powder in liquid nitrogen. Total proteins were extracted in RIPA buffer, and quantified by BCA assay. For the indicated ages, an equal amount of proteins (that depended on the expression level of Bcl-xL in each tissue) was separated on tris-taurine-glycine minigels. Immunodetection of Bcl-x was performed, and revealed 3 bands: unmodified BCI-XL (U), N52 monodeamidated BCI-XL (M) and doubly deamidated BCI-XL (D).

Figure 2. A-D. The effect of Metformin and Rapamycin on the relative amount of deamidated Bcl-xL.

EXAMPLE:

Firstly, it is remarkable that fetal cells express Bcl-xL under its native form, and that monodeamidation only appears after birth (see Figure 1). Bcl-xL deamidation profile can be used as a readout for cellular primordial youth. It can also be used as a readout for the efficiency a given compound might have to slow down aging, or to rejuvenate cells.

Secondly, HeLa cells were treated for the indicated times with 100 nM of the indicated anti-aging compounds (the compounds where refreshed when the cells were passaged every 4 days). Whole proteins were extracted in RIPA, protein concentration was determined and 20 pg were separated by SDS-PAGE, followed by immunodetection of Bcl-xL. Native and N52- monodeamidated Bcl-xL were quantified by densitometric analysis followed by a deconvolution of the signal as in Beaumatin et al., Oncotarget 2016). The effect of Metformin and Rapamycin on the relative amount of deamidated Bcl-xL was plotted (see Figures 2A-2D).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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6. Szymanska, G., Leszyk, J. D. & O’Connor, C. M. Carboxyl Methylation of

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7. Wright, H. T. Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins. Protein Eng. 4, 283-294 (1991).

Banaszynski, L. A., Chen, L.-C., Maynard- Smith, L. A., Ooi, A. G. L. & Wandless, T.

J. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995-1004 (2006).

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Claims

CLAIMS:

1. A fusion protein wherein the Bcl-xL protein is fused to a stability-affecting protein.

2. The fusion protein of claim 1 wherein the stability-affecting protein is a variant of the FKBP12 protein.

3. The fusion protein of claim 2 wherein the stability-affecting protein is the mutant L106P of FKBP12.

4. The fusion protein of claim 1 wherein the stability-affecting protein has an amino acid sequence as set forth in SEQ ID NO:3.

5. An isolated, synthetic or recombinant nucleic acid molecule encoding for the fusion protein of claim 1.

6. A vector comprising the nucleic acid of claim 5.

7. A host cell which has been transfected, infected or transformed by the nucleic acid molecule of claim 5 and/or the vector of claim 6.

8. A non-human transgenic animal harbouring engineered alleles that direct the expression of the fusion protein of claim 1.

9. Use of the host cell of claim 7 or the non-human transgenic animal of claim 8 for screening anti-aging agents and/or rejuvenating agents.

10. A method for identifying a substance useful as anti-aging agent comprising the steps of i) providing a host cell or a non-human transgenic animal, ii) contacting said host cell or non-human transgenic animal with an amount of the stabilizing ligand for a sufficient time for allowing the expression of the fusion protein, iii) withdrawing the stabilizing ligand for a sufficient time for allowing the degradation of the fusion protein, iv) contacting the host cell or non-human transgenic animal with an amount of a test substance in the presence of an amount of the stabilizing ligand for a sufficient time for allowing the expression of the fusion protein v) performing a time-course analysis to monitor the rate of mono-deamidation of fusion protein in which the ability of the test substance to act as an anti-aging agent is indicated by the slowdown detection of the mono-deamidated form as compared to the deamidation rate observed in the absence of the test substance and (vi) selecting the substance that slowdown the apparition of the mono-deamidated form.

11. A method for identifying a substance useful as rejuvenating agent comprising the steps of i) providing a host cell or a non-human transgenic animal, ii) contacting said host cell or non-human transgenic animal with an amount of the stabilizing ligand for a sufficient time for allowing the expression of the fusion protein and reaching a pre-treatment steady state of deamidation, iii) withdrawing the stabilizing ligand for a sufficient time for allowing the degradation of the fusion protein, iv) contacting the host cell or non human transgenic animal with an amount of a test substance in the presence of an amount of the stabilizing ligand for a sufficient time to allow the expression of the fusion protein and to reach a post-treatment steady state v) comparing the steady state of deamidation of the fusion protein before and after exposure to the test substance; whereby the rejuvenating effect of the substance is indicated by the decreased accumulation of the mono-deamidated form after treatment as compared to the deamidation steady state observed before treatment and finally (vi) selecting the substance that resets the epigenetic clock of the cell, ie rejuvenate cellular parameters enough to impair the accumulation of the mono-deamidated form.

12. The method of claims 10 or 11 wherein the stabilizing agent is shieldl.

13. A kit comprising an amount of fusion proteins and/or nucleic acid molecules and/or host cells.

14. The kit of claim 13 which further comprises an amount of stabilizing ligands.

15. The kit of claim 14 wherein the wherein the stabilizing agent is shieldl.