FR2925314A1 - Use of transglutaminase 3 or related substances to prevent or treat signs of skin aging - Google Patents

Abstract

Transglutaminase 3 (I) or a fragment of (I) or a nucleic acid encoding (I) or a modulator of the activity or expression of (I) is used cosmetically to prevent or treat signs of skin aging or to prepare a therapeutic composition for this purpose. The polypeptide (I) encoded by SEQ ID NO:1 (defined sequence of 2391 nucleotides given in the specification) or a fragment of (I) or a nucleic acid encoding (I) or a modulator of the activity or expression of (I) is used cosmetically to prevent or treat signs of skin aging or to prepare a therapeutic composition for this purpose. Independent claims are also included for: (1) use of (I) to screen for compounds capable of modulating the expression or activity of (I); (2) characterizing the condition of epithelial tissue by determining the amount of (I) or (I)-encoding nucleic acid in a tissue sample and comparing the result with a reference value; (3) screening for antiaging compounds by contacting a (I)-expressing cell with a test compound and determining the amount of (I) expressed; (4) characterizing the efficacy of a cosmetic or therapeutic treatment intended to compensate for signs of skin aging by qualitatively or quantitatively characterizing the expression or biological activity of (I); (5) use of (I) or a modulator of the expression of (I) to prepare or improve a multilayer cellular model, especially a reconstructed skin model.

Description

The present invention relates to the use, in particular cosmetic and / or therapeutic, of transglutaminase 3, of polypeptides derived from this protein, for example derived from its proteolysis, or analogs thereof, of a nucleic sequence. coding for such a polypeptide or an agent modulating the activity or the expression of such a polypeptide, in particular for preventing and / or treating the signs of cutaneous aging. The invention also relates to the use of transglutaminase 3, polypeptides derived therefrom or analogs thereof, or a nucleic sequence encoding such polypeptide as a marker for the evaluation of a state of an epithelium, and especially of the epidermis. Epithelia are tissues whose cells are contiguous and integral with one another and rest on a basement membrane. They form an outer coating, for example on the surface of the skin, or the epidermis, or internal, on the surface of a mucosa. They can also form glands.

More specifically, these epithelia are structures whose homeostasis results from the implementation of a finely regulated set of intracellular and extracellular signals acting at all stages of proliferation, migration, cell differentiation and than the synthesis of the different components of the extracellular matrix. These signals may, in particular, result from the action of factors produced by keratinocytes. Maintaining the good physiological functions of an epithelium involves, in particular, terminal epithelial differentiation and / or proteoglycan synthesis. As regards more particularly the epidermis, it is an epithelium, conventionally divided into a basal layer of keratinocytes containing, in particular, cutaneous stem cells and constituting the germinal layer of the epidermis, a so-called thorny layer constituted several layers of polyhedric cells disposed on the basal layer, a so-called granular layer comprising one to three layers of flattened cells containing distinct cytoplasmic inclusions, keratohyaline grains, and finally, a set of upper layers, called the horny layer (or stratum corneum) consisting of end-stage keratinocytes of their differentiation called corneocytes.

The stratum corneum, the outermost part of the skin that provides the barrier function between the body and the environment, and the hair shaft, the emergent part of the hair follicle that makes up the hair, are both the culmination of the healing process. differentiation of keratinocytes. Epidermal differentiation follows a maturation process in which basal layer keratinocytes differentiate and migrate to form corneocytes, dead cells that are completely keratinized. This differentiation is the result of perfectly coordinated phenomena that will lead to the maintenance of a constant thickness and thus ensure the homeostasis of the epidermis.

Many disorders or skin diseases can result from a dysfunction of the homeostasis of the epidermis. For example, in the case of aged skin, this dysfunction is generally manifested by the appearance of wrinkles (micro-relief and deep wrinkles), a loss of elasticity, a rough feel and dryness. Histologically, a flattening of the dermal-epidermal junction and a decrease in thickness of the dermis and epidermis are observed. The content of collagen and glycosaminoglycans decreases. The barrier function of the skin is impaired. All of these phenomena are increased by chronic sun exposure. Likewise, this dysfunction may be exacerbated in women during menopause. The present invention results particularly from the characterization by the inventors of the expression of the transglutaminase 3 protein in the stratum corneum of the human epidermis, in particular in a dry and aged human epidermis. Transglutaminases are calcium-dependent enzymes that catalyze cross-linking of structural proteins by formation of epsilon-gamma-glutamyl-lysine interchain covalent bridges between lysine and glutamine residues of adjacent proteins, generating an insoluble macromolecular aggregate (Folk et al. Adv., Prot Chem 31: 1-133, 1977). Transglutaminase 3 (also known as glutamine-gamma-glutamyltransferase protein) is a heterodimeric protein whose activation requires the cleavage of a precursor form comprising 693 amino acids (SEQ ID NO 2) and has a molecular weight of about 77 kDa. . The heterodimer consists of a catalytic subunit of 223 amino acids (27 kDa) and a non-catalytic subunit of 470 amino acids (50 kDa) through a transcriptomic analysis performed on human prepuces , Lener et al. (Gerontol Exp., 2006 Apr., 41 (4): 387-97) observed a decrease in the expression of mRNA encoding transglutaminase 3 in an elderly individual compared to a young individual. Transglutaminase 3 is expressed in the final stages of terminal differentiation of the epidermis and in certain types of hair follicles. This enzyme appears to play an important role in the formation of the horny envelope of cells, contributing in particular to the rigidity of its structure, one of the essential mechanisms to ensure the barrier function between the body and the environment. Thus, the present invention results more particularly from the unexpected observation by the inventors of a decrease in the expression of the transglutaminase 3 protein in an aged group relative to a younger group, specifically in the stratum corneum. Indeed, contrary to all expectations, transglutaminase 3 proves to be also a potential marker of the physiological state of the skin, particularly in terms of aging. Accordingly, according to one of its first aspects, the subject of the present invention is a cosmetic or non-therapeutic use of an effective amount of at least one polypeptide derived from transglutaminase 3 and, in particular, from an amino acid sequence. encoded by a nucleic acid sequence represented in whole or in part by a sequence represented by SEQ ID NO 1, an analogue thereof or a fragment thereof, of at least one nucleic sequence encoding such a polypeptide or at least one agent that modulates the activity or the expression of such a polypeptide as a useful agent for preventing and / or treating the signs of cutaneous aging. According to another of its aspects, the subject of the present invention is also a use of an effective amount of at least one polypeptide derived from transglutaminase 3 and, in particular, from an amino acid sequence encoded by a nucleic acid sequence. represented in whole or in part by a sequence represented by SEQ ID NO 1, an analogue thereof or a fragment thereof, at least one nucleic sequence coding for such a polypeptide or at least one the activity or the expression of such a polypeptide for the preparation of a composition, in particular a therapeutic composition, intended to prevent and / or treat the signs of cutaneous aging. For the purposes of the present invention, the term "effective amount" is intended to denote the minimum amount necessary for observing the expected effect, namely a cosmetic effect or a therapeutic effect, it being understood that the effective amounts required for obtaining a cosmetic effect or a therapeutic effect may be, where appropriate, the same or different. For the purposes of the invention, the term "cosmetic use" denotes a use intended primarily to provide an aesthetic effect and / or comfort.

Within the meaning of the invention, the term "therapeutic composition" is intended to denote a composition intended to provide a prophylactic or curative effect with regard to epithelial, and in particular epidermal, disorders recognized as reflecting a pathological state. By prophylactic or preventive in the sense of the invention is meant the reduction of the risk of occurrence of a phenomenon, for example a pathology. A composition according to the invention may, in particular, be intended to prevent and / or treat a thinning of an epidermis and / or a loss of firmness, elasticity, density and / or tonicity of an epithelium, in particular an epidermis and / or the signs of skin aging, such as the formation of wrinkles and fine lines.

By signs of cutaneous aging is meant all the changes in the external appearance of the skin due to aging whether chronobiological and / or photoinduced, such as, for example, wrinkles and fine lines, withered skin, lack of elasticity and / or tone of the skin, thinning of the dermis and / or degradation of collagen fibers resulting in the appearance of soft and wrinkled skin.

According to another embodiment, a composition according to the invention may, in particular, be intended to prevent and / or treat cutaneous signs of dryness, in particular to prevent and / or treat dehydration of an epidermis. A composition according to the invention may, in particular, be intended to prevent and / or treat the effects of the chronological aging of an epithelium, in particular an epidermis or the lips or the scalp. According to another aspect, the present invention also relates to the use of at least one polypeptide in accordance with the invention as a screening tool for biological or chemical compounds capable of modulating, and in particular of inhibiting, expression and / or the biological activity of said polypeptide. In particular, it relates to a method for screening anti-aging active agents comprising at least the steps of: a) bringing into contact, at least one cell type capable of expressing a polypeptide according to the invention, that is to say expressing the transglutaminase 3 or one of its derivatives with at least one chemical or biological compound to be tested, under conditions favorable to a manifestation of the expression of said polypeptide, and b) determining a content of said polypeptide.

According to yet another of its aspects, the present invention also relates to the use of at least one polypeptide according to the invention, or at least one nucleic acid sequence coding for said polypeptide, as a tool for in vitro or ex vivo characterization of a state of an epithelium, and in particular of an epidermis. More precisely, the present invention relates, according to another of its aspects, to a method, in particular a cosmetic, non-invasive method for characterizing the surface state of an epithelium, in particular an epidermis, comprising at least the qualitative or quantitative characterization. of the expression and / or the biological activity of a polypeptide according to the invention, that is transglutaminase 3, of one of its derivatives or fragments. According to an alternative embodiment, the datum or value obtained can be appreciated in comparison with a datum or reference value, obtained for example from at least one epithelium, in particular an epidermis, distinct from that which is the subject of the characterization, and whose state is known. According to another of its aspects, the present invention also aims at a process, in particular a cosmetic, non-invasive method for characterizing the effectiveness of a cosmetic or therapeutic treatment intended to supplement the signs of cutaneous aging, comprising at least the qualitative or quantitative characterization of the skin. the expression and / or the biological activity of a polypeptide according to the invention, that is to say transglutaminase 3, one of its derivatives or fragments. According to an alternative embodiment, the data obtained at the end of the characterization can also be examined compared to a reference value or datum. This value or reference datum may be data obtained from the epithelium, in particular from the epidermis, to be subjected to treatment, prior to the administration of said treatment or in a shorter chronological time with respect to the start date. treatment. As is apparent from the description which follows, the methods according to the invention are particularly advantageous insofar as their implementation does not require an invasive operation. The methods of the invention can be carried out in vitro, ex vivo or in vivo. In fact, the location by the inventors of the new aging biomarker transglutaminase 3 in the stratum corneum makes it possible to characterize quantitatively or qualitatively the expression of this protein by simple topical sampling. The sampling method may for example be a stripping type of technique of applying to the epithelium considered, such as an epidermis, a portion of adhesive tape. By detaching this adhesive tape, a fraction of the epithelium, for example an epidermal fraction, is removed. After protein extraction, it is then analyzed by conventional methods, such as the enzyme immunoassay or more particularly a Western-Blot analysis.

POLYPEPTIDE DEFINITION According to one embodiment, a polypeptide that is suitable for the invention may have an amino acid sequence represented in whole or in part by a sequence represented by SEQ ID NO 2, or an analogue thereof, or a fragment of it. For the purposes of the present invention, the term "transglutaminase 3" is generally used to denote, unless otherwise indicated, the sequence (SEQ ID NO 2) of the protein, whether or not it has undergone post-translational modifications, of the enzymatic cleavage type between the residues. amino acids 470 and 471, which may change its apparent molecular weight or isoelectric point. It is moreover known that the primary sequence of a polypeptide, that is to say the sequence of amino acids, determines sites specifically recognized by protease-type enzymes, such as trypsin which, once recognition is obtained. of these effective sites will induce cleavage of the polypeptide by proteolysis. This proteolysis results in the generation of various peptides, or fragments of proteolysis, of transglutaminase 3. The inventors have detected the presence of such peptides in the stratum corneum.

Accordingly, the invention also extends to the proteolysis fragments of transglutaminase 3. Thus, according to a particular embodiment, a polypeptide that is suitable for the invention may have an amino acid sequence chosen from 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, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, and mixtures thereof. The term "polypeptide analogue" is intended to denote any polypeptide having a sequence homology, in particular with respect to one of the characteristic sequences of said polypeptide, as well as a biological activity of the same kind. This analog may be a peptidomimetic agent.

The homology can be at least 85%, for example at least 90%, and for example at least 95%. The homology can be determined by visual comparison or by any computer tool commonly used in the field such as BLAST programs available at www.ncbi.nlm.nih.gov and used with the default settings.

The sequence homology may result from modifications resulting from mutation or variation in the sequences of the peptides according to the invention either from the deletion or insertion of one or more amino acids or from the substitution of one or more amino acids. several amino acids in the characteristic sequences of a polypeptide according to the invention. Within the meaning of the invention, the term "polypeptide fragment" is intended to denote any portion of a polypeptide according to the invention comprising at least 4, at least 6, in particular at least 8, and more particularly at least 12 consecutive amino acids of said polypeptide. polypeptide, and a substantially similar biological activity. The term "characteristic sequence of the polypeptide" is intended to refer in particular to transglutaminase 3, the sequence represented by SEQ ID NO 2.

In general, the polypeptide analogs may comprise conservative substitutions with respect to the amino acid sequence of the native polypeptide.

Many of these changes can be combined. By way of example of mutations which may be considered in the present invention, it may be mentioned, in a non-exhaustive manner, the replacement of one or more amino acid residues by amino acid residues having a subscript. similarly without substantially affecting the biological properties of the polypeptide, and in particular its biological activity such as its activity of stimulating the proliferation and / or the migration and / or the terminal differentiation of keratinocytes or the stimulation of the synthesis of proteoglycans at the level of an epithelium, and in particular of the epidermis.

The hydropathic index is an index attributed to amino acids as a function of their hydrophobicity and charge (Kyte et al (1982) J. Mol Biol., 157: 105). A polypeptide or the like also targeted by the present invention may be a polypeptide having undergone one or more post-translational modification (s). By post-translational modification (s), it is meant to encompass all the modifications that a peptide or a protein is likely to undergo after its synthesis in a cell, such as, for example, one or more phosphorylation. (S), one or thiolation (s), acetylation (s), glycosylation (s), lipidation (s), such as farnesylation or palmitoylation, a structural rearrangement of the type formation of disulfide and / or cleavage bridges within the peptide sequence. The analogue has, moreover, substantially the same biological activity as the natural polypeptide. According to one embodiment, a polypeptide that is suitable for the implementation of the invention may also be a natural or synthetic polypeptide, which may optionally be obtained after enzymatic or chemical lysis of transglutaminase 3 or by chemical or biological synthesis or by extraction from a biological tissue, such as the skin, naturally expressing this polypeptide or after transfection thereof, as well as the various post-translational forms thereof, or any natural or synthetic polypeptide whose sequence comprises wholly or partially (in whole or in part) a aforementioned amino acid sequence, for example variants and analogs.

Those skilled in the art can obtain a polypeptide according to the invention by means of recombinant DNA methods, for example, those described in the manual Molecular Cloning - A Laboratory Manual (2nd Edition), Sambrook et al. ., 1989, Vol. I-III, Coldspring Harbor Laboratory, Coldspring Harbor Press, NY, (Sambrook). According to another embodiment, a polypeptide suitable for the implementation of the invention may also be a polypeptide as defined above in which at least one residue has been replaced by an amino acid residue of similar hydropathic index, as defined previously.

According to another embodiment, a polypeptide suitable for the implementation of the invention may also be a polypeptide as defined above, fused with another polypeptide, a hydrophilic or hydrophobic targeting agent, a bio-conversion precursor, an agent luminescent, radioactive or colorimetric marking. In a nonlimiting manner, examples of compounds that may be coupled with a polypeptide according to the invention include fluorescent proteins such as Green Fluorescent Protein, fluorescent chemical compounds such as rhodamine, fluorescein, or Texas Red. , phosphorescent compounds, radioactive elements, such as 3H, 14C 35S 1211 or 125I, or colorimetric labeling agents such as chromogenic substrates sensitive to the action of galactosidase, peroxidase, chloramphenicol acetyltransferase, luciferase or alkaline phosphatase. Depending on the nature of the compounds which can be coupled with a polypeptide according to the invention, the coupling can be carried out by chemical methods, in particular by means of reactive chemical functions or by methods of molecular biology known to humans. 'art.

DEFINITION OF NUCLEIC ACID SEQUENCES According to one embodiment, the present invention also relates to nucleic acid sequences coding for a polypeptide of the invention and to their use in the various uses and processes in accordance with the invention. Thus, the present invention also relates to the use of nucleic acid sequences, in particular of deoxyribonucleic acids, or of ribonucleic acids encoding a polypeptide according to the invention, in particular the sequences corresponding to at least one sequence of nucleic acids represented by SEQ ID NO: 1, analogs thereof or a fragment thereof for the preparation of a composition according to the invention.

For the purposes of the present invention, the term "nucleic acid sequence fragment" is intended to denote a nucleic acid sequence encoding all or part of a polypeptide according to the invention, or an analogue thereof, and particularly a nucleic acid sequence represented by SEQ ID NO 1 or an analogue thereof.

By analog of a nucleic acid sequence is meant any nucleic acid sequence, possibly resulting from the degeneracy of the nucleic acid code, and coding for a polypeptide of identical or analogous sequence to that of the polypeptide encoded by said sequence nucleic acids. The nucleic acid sequences can come from all possible origins, namely either animal, in particular mammalian and even more particularly human, or plant, or microorganisms (viruses, phages, bacteria among others) or fungi , without prejudging the fact that they are present naturally or not in said original organism. In this case, the invention also relates to the use of isolated and purified nucleic acid fragments coding for the polypeptides considered according to the invention. A nucleic acid sequence according to the invention may comprise a sense, antisense or interferential sequence corresponding to a sequence coding for a polypeptide according to the invention.

Thus, the present invention also relates to the use of nucleic acid sequences, especially deoxyribonucleic acids, or ribonucleic acids encoding a polypeptide according to the invention. The nucleic acid sequences according to the invention may in particular be used to prepare the corresponding ribonucleic acid sequences, sense or antisense. The subject of the invention is also the use of any polynucleotide, of ribonucleic or deoxyribonucleic acid sequence, comprising a sense or antisense sequence, in particular Small interferent RNA (SiRNA), corresponding at least to the nucleic acid sequence SEQ ID NO 1 or an analogue thereof.

MODULATOR AGENT According to another embodiment, the invention relates to the use of a modulating agent for the expression and / or the activity of a polypeptide according to the invention. By modulating agent of transglutaminase 3 is meant a product capable of interfering with the enzymatic activity thereof. The modulator may indirectly interfere by altering the amount of endogenous enzyme produced, including promoting or, conversely, decreasing its gene expression and / or translation. Thus, any agent modulating the gene or protein expression of transglutaminase 3 may be suitable for the invention. The modulating agent may also interfere directly with the enzymatic reaction catalyzed by transglutaminase 3. These are for example enzymatic activators or inhibitors. In particular, the invention relates to the use of a modulating agent activating the expression of transglutaminase 3. By way of illustration and not limitation of such activating agents, mention may in particular be made of agents capable of mobilizing intracellular calcium, transcription factors Sp 1 and Ets, or compounds promoting keratinocyte differentiation, such as vitamin D and its derivatives, histone deacetylase inhibitors such as butyrate, PPARgamma agonists. According to a preferred embodiment, the modulating agent is an enzymatic activator.

By way of nonlimiting illustration, such enzymatic activators may be chosen from the cathepsin D protease, responsible for the cleavage of the precursor form of the enzyme, sphingosine phosphorylcholine, CLSP or certain fragments of this protein, GTP analogs. (Guanosine Tri Phosphate), or agents capable of increasing the intracellular concentration of calcium, such as thapsigargin.

By biological activity is meant in particular with regard to transglutaminase 3, the biological activity of the protein represented by the sequence SEQ ID No. 2, as well as the cleaved mature form.

The present invention further relates to a method for screening biological or chemical compounds or physicochemical factors capable of modulating a biological activity of a polypeptide according to the invention comprising at least the steps of: a) bringing into contact with at least one chemical or biological compound to be tested, at least one polypeptide according to the invention and / or subjecting said polypeptide to said physicochemical factor, under conditions conducive to the manifestation of said biological activity of said polypeptide, and b) determine said biological activity of said polypeptide.

In such a method, the biological activity of the polypeptide, in particular its epithelial differentiation activity, and in particular epidermal terminal differentiation, especially with regard to keratinocytes, may be determined by any method known to those skilled in the art. For example and in a nonlimiting manner, mention may be made of cell culture methods followed by characterization of differentiation markers, such as, for example, keratin 10, filaggrin or proliferation markers, such as, for example, KI 67 and PCNA . According to one embodiment, the biological activity of the polypeptide can be compared to a reference value.

A reference value can be obtained by measuring the biological activity of the polypeptide in the absence of any biological or chemical compound or physicochemical factor to be tested. Assuming that this reference value measurement is carried out prior to the use of the biological or chemical compound or the physicochemical factor to be tested, the method according to the invention can also, if necessary, make it possible to assess the potential efficacy of said compound. This biological activity may not be affected by the presence of said compound or on the other hand be inhibited or stimulated. In the event that an inhibitory effect is found, the test compound is likely to be used for example as an anti-aging active. A method according to the invention can be carried out on an isolated cell sample, obtained either from a skin biopsy or from cells in culture.

Advantageously, as cell sample suitable for the invention, there may be mentioned a sample of keratinocytes. Advantageously, a polypeptide used in a process according to the present invention may be transglutaminase 3.

The present invention also relates to a method for screening biological or chemical compounds capable of modulating the expression of a polypeptide according to the invention comprising at least the steps of: a) bringing into contact at least one cell type capable of expressing a nucleic acid sequence coding for said polypeptide according to the invention with at least one chemical or biological compound to be tested, under conditions favorable to the expression of said sequence, and b) determining the expression of said nucleic acid sequence. The expression of a nucleic acid sequence can be determined, for example, by means of oligonucleotide probes, by any protocol known to those skilled in the art. By way of example of nucleic acid sequence detection methods, mention may be made of the polymerase chain reaction (PCR), quantitative (Q-PCR) or not, in the presence or absence of reverse transcriptase ( RT-PCR or Q-RT-PCR), Northern blot, ribonuclease protection assay method, microarray methods, transcriptomic chip methods, oligonucleotide microarray methods, in situ hybridization. By way of example of agents suitable for the detection of a nucleic acid sequence, and in particular mRNA, mention may be made of labeled nucleic acid probes capable of hybridizing to said sequence. Such a nucleic acid probe can be easily obtained by any method known to those skilled in the art. Thus, the nucleic acid sequences according to the invention can be used to produce sense and / or antisense oligonucleotide primers which hybridize under high stringency conditions to the sequence SEQ ID No. 1 or a similar of it.

The expression of a nucleic acid sequence according to the invention may be compared with a reference value obtained, for example, by carrying out a process according to the invention in the absence of test compound. The expression of a nucleic acid sequence can also be determined, indirectly, by determining the expression of the polypeptide encoded by said sequence, by means of any technique known in the art, such as Western Blot. , the ELISA, the Bradford or Lowry method, or as indicated below. The present invention also relates to a method for screening biological or chemical compounds, or even anti-aging active agents, capable of modulating the expression of a polypeptide according to the invention, comprising at least the steps of: a ) contacting at least one cell type capable of expressing a polypeptide compliant with at least one chemical or biological compound to be tested, according to the invention, under conditions favorable to the manifestation of the expression of said polypeptide. b) determining a content of the polypeptide, and c) comparing said content determined in step b) with a content of said determined polypeptide in the absence of chemical or biological compound to be tested. The comparison made in step c) may make it possible to deduce information as to the property of said test compound to modulate the expression of a polypeptide according to the invention. A process according to the invention can be carried out on an isolated cell sample. Determination of a polypeptide content according to the invention can be carried out using any method known to those skilled in the art.

As methods of detecting a polypeptide, mention may be made of Western blot, blot-blot, Dot-blot, single-gang or multiplex Enzyme Linked Immuno-Sorbent Assay (ELISA) methods, proteomics or glycomics, staining polypeptides in a polyacrylamide gel with a silver dye, Coomassie blue or SYPRO, immunofluorescence, UV absorption, immunohistochemical methods in microscopy classical, electronic or confocal, FRET (fluorescence resonance energy transfer), TR-FRET methods (time resolved FRET / FRET), FLIM (fluorescence lifetime imaging) methods microscopy / fluorescence time microscopy imaging), FSPIM methods (fluorescence spectral imaging microscopy), FRAP methods (fluorescence recovery after phot fluorescence recovery / recovery after photobleaching), reporter gene methods, atomic force microscopy (AFM) methods, surface plasmon resonance methods, microcalorimetry methods, flow cytometry methods , biosensor methods, radioimmunoassay (RIA) methods, isoelectric focusing methods, and enzymatic assays, methods using peptide chips, sugar chips, antibody chips, methods of mass spectrometry, SELDI-TOF spectrometry methods (Ciphergen). The methods according to the invention can be carried out on a sample, for example isolated, of epithelium, in particular of epidermis, obtained from a cutaneous biopsy or an epithelial cell model, for example epidermal or more advantageously a non-invasive surface removal, in particular by stratum corneum tape or by simple washing. Sampling of an epidermis sample can be done by any method known to those skilled in the art.

These methods can be performed by so-called stripping techniques. These strippings are tacky surfaces applied to the surface of the epidermis such as 3M's Blenderm, D'squam (commercial adhesive from CuDERM), cyanoacrylate glue or the varnished stripping method. Thanks to these strippings, the adherent corneocytes and the contents of their intercellular spaces can be removed and subsequently subjected to an extraction allowing access to the protein content. The sampling of a sample suitable for the process can also be carried out more directly by washing the skin surface by means of, for example, finned turbine type spiral cell type accessories (as described in the FR 2 patent). 667 778) associated with a fluidic circuit, or simply by adding / removing a drop of buffer on the surface of the skin. As an indication, other sampling methods adapted to the implementation of the invention may be mentioned, such as scraping methods of the upper part of the stratum corneum by means of a bimetallic system. This technique allows the collection of scales that can then be directly analyzed by different techniques to determine the levels of minerals, amino acids or lipids. It is understood that all of the cosmetic or therapeutic compositions considered according to the invention use a physiologically acceptable medium. For the purpose of the present invention, the term "physiologically acceptable medium" is intended to mean a medium that is suitable for applying a composition to an epithelium or a keratin material, such as the skin, the scalp, the lips, the mucous membranes and the skin. keratinous fibers such as hair, nails and hair, or where appropriate orally or parenterally. Within the meaning of the present invention, the term "treatment" designates a composition that can be used as part of a prophylactic and / or curative treatment, or a method for evaluating a state of an epithelium, and especially of the epidermis.

According to another embodiment, a cosmetic or therapeutic composition according to the invention may comprise, in addition, at least one cosmetic and / or therapeutic active agent. As examples of active agents that may be used in the context of the present invention, mention may be made of cosmetic oils, such as silicone oils, vegetable oils of the triglyceride type, hydrocarbon oils such as Parleam oil and esters of fatty acids and fatty alcohol. It may also be possible to use other active agents which make it possible to improve the state of the skin, such as moisturizing or moisturizing active agents or active agents which make it possible to improve the natural lipid barrier, such as ceramides, cholesterol sulphates and / or fatty acids, and mixtures thereof. It may also be possible to use enzymes having activity on the skin, such as proteases, lipases, glucosidases, amidases, cerebrosidases and / or melanases, and mixtures thereof. Other examples of active agents that are suitable for carrying out the present invention include: analgesic active agents, anti-yeast active agents, antibacterial active agents, anti-parasitic active agents, anti-fungal active agents, anti-viral active agents steroidal anti-inflammatory active agents, anesthetic active agents, anti-pruriginous active agents, keratolytic active agents, anti-free radical active agents, anti-seborrhoeic active agents, anti-dandruff active agents, anti-acne active agents, active ingredients to prevent aging of the skin and / or to improve its condition, anti-dermatitis active agents, anti-irritant active agents, immunomodulatory active agents, active ingredients for the treatment of dry skin, antiperspirant active ingredients, anti-active ingredients -specific, UV protective active ingredients, anti-histamine active ingredients, healing active ingredients, self-tanning active ingredients, antioxidants such as green tea or active fractions thereof ci, glycerine, laponite, caffeine, aromatic essential oils, dyes, depigmenting active ingredients, liporegulators, softening, refreshing, deodorant, numbing, whitening, nourishing, active ingredients that reduce differentiation and / or proliferation and / or skin pigmentation, and mixtures thereof. In general, any composition of the invention can be applied to the skin (on any cutaneous zone of the body) or on the mucous membranes (oral, jugal, gingival, genital, conjunctival, ...).

In a known manner, a cosmetic composition may also contain adjuvants customary in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillers, filters, odor absorbers and dyestuffs.

The amounts of the various constituents of the compositions according to the invention are those conventionally used in the fields under consideration. The amount of chemical or biological compound or polypeptide, nucleic acid sequence or modulating agent according to the invention contained in a composition according to the invention, also known as the effective amount, is of course dependent on the nature of the compound and the desired effect and may therefore vary to a large extent. To give an order of magnitude, a composition may contain a modulating agent according to the invention or a polypeptide in an amount representing from 0.00001% to 50% of the total weight of the composition, in particular in an amount representing 0.001% at 10% of the total weight of the composition, and more particularly in an amount representing from 0.1% to 1% of the total weight of the composition.

A composition according to the invention may be more particularly dedicated to the reduction and / or treatment of conditions likely to deteriorate the state of an epithelium, and in particular of an epidermis. Such a state may be of chronological origin (i.e. related to the elapsed time as cutaneous aging) and / or indicative of a skin disorder, linked for example to photoaging. Thus, a composition according to the invention, in particular a cosmetic one, may, in particular, be intended to prevent and / or treat a thinning of an epidermis and / or a loss of firmness, elasticity, density and / or tonicity of an epidermis and / or the formation of wrinkles and fine lines. According to another embodiment, a composition according to the invention, in particular cosmetic, may, in particular, be intended to prevent and / or treat cutaneous signs of dryness, in particular to prevent and / or treat a dehydration of the skin. an epidermis.

According to another embodiment, a composition according to the invention, in particular cosmetic, may be intended to prevent and / or treat signs of epidermal aging. A composition according to the present invention, in particular therapeutic, may be more particularly intended for the treatment of a skin disorder such as a disorder of the hydration of the skin, such as xerosis, parakeratosis, hyperkeratosis, ichthyosis, psoriasis, atopic dermatitis, eczema, rosacea, lichen, pruritus, skin pathology with an inflammatory component or resulting from an alteration of the immune response, desquamation, dysregulation of melanogenesis or sebogenesis, alopecia, hirsutism, a healing disorder, or a skin disorder involving secretory phenomena and the process of cellular invasion, particularly in the context of malignant or benign neoplasias. In another aspect, the present invention also relates to the use of at least one polypeptide according to the invention or at least one nucleic acid sequence encoding said polypeptide, as a characterization tool of a state of an epithelium, and especially of an epidermis. By way of example, it is possible to characterize according to the invention a state of an epithelium chosen from the dryness of an epidermis, chronological aging and photoaging. Thus, as specified above, the present invention relates, according to another of its aspects, to non-invasive methods for characterizing the surface state of a non-pathological epidermis or the effectiveness of a cosmetic or therapeutic treatment aimed at characterizing qualitatively or quantitatively the expression of transglutaminase 3, one of its derivatives or fragments. These methods are particularly advantageous insofar as their implementation does not require mandatory use of an operative technique to carry out such a characterization. An extract of the epidermis can thus be obtained by simple stripping and directly analyzed by a conventional analysis technique, especially as described above. According to one embodiment, a method of characterizing a state of an epithelium, for example an epidermis, comprises at least the steps of: a) determining, in a sample of said epithelium, a content of a polypeptide conforming to the invention, or a nucleic acid sequence encoding said polypeptide, and b) comparing said content determined in step a) to a reference value. Advantageously, a method of the invention is non-invasive. A method of the invention is advantageously carried out on an isolated sample. According to one embodiment, a method according to the invention can be carried out on a sample of epithelium, and in particular of epidermis, taken from an individual. A method according to the invention may also be carried out on a sample of epithelium, and in particular of epidermis, taken from an epithelial cell model, and in particular an epidermal cell model, or from an isolated reconstructed skin in order to describe it. the state. The sampling of an epithelium sample can be done by any method known to those skilled in the art. A method according to the invention can be carried out in vivo, in vitro or ex vivo. A reference value may be, for example, a polypeptide or nucleic acid sequence content determined on a sample of epidermis taken from an epithelium, and in particular normal skin, that is to say satisfactory on the skin. physiological plan, like for example a young skin.

The measurement of a reference value may be performed in parallel or sequentially to the determination of said content of a polypeptide or nucleic acid sequence. A comparison of a determined content with a reference value may make it possible to evaluate a deviation from this value. The analysis of the intensity and / or the nature of this deviation (negative or positive) can be informative of the condition of the epidermis. Characterization of a condition of an epidermis may be indicative of a possible skin disorder that can be corrected by the use of compounds capable of modulating the expression of a polypeptide of the invention. According to one embodiment, a method according to the invention can be implemented in a method of in vivo, in vitro or ex vivo diagnosis of aging of an epithelium, and in particular of the epidermis, in an individual. A polypeptide suitable for carrying out a process according to the invention may advantageously be transglutaminase 3. The determination of a content of polypeptide according to the invention or of nucleic acids according to the invention in a sample of The epidermis may be performed by any protocol known to those skilled in the art. As methods of detecting a polypeptide, mention may be made of those mentioned above. Thus, it may be envisaged to detect the presence of a polypeptide according to the invention by means of an antibody, where appropriate in a marked form. An antibody that can be used as a skin condition assessment tool can be obtained by any method known to those skilled in the art, as described in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). Advantageously, the antibodies used may be recombinant antibodies such as those developed by Antibodies-by-design. A nucleic acid sequence suitable for carrying out a method according to the invention may advantageously be a nucleic acid sequence coding for transglutaminase 3, for example of the mRNA type. As an example of methods for detecting nucleic acids in accordance with the invention, mention may be made of the methods mentioned above.

The present invention also relates to a method, non-therapeutic, for demonstrating an effect of a treatment likely to regress a disorder of an epithelium, especially a skin disorder or scalp, in an individual, comprising at least the steps comprising: a) performing, before the treatment, at least a first determination, in a first sample of an epithelium taken from said individual, of a biological activity and / or the expression of a polypeptide conforming to the invention, or expression of a nucleic acid sequence encoding said polypeptide; b) performing, after the treatment, at least a second determination, in a second sample of an epithelium taken from said individual, of said activity; and / or said expression of said polypeptide or said expression of said nucleic acid sequence determined in step a), and c) comparing the first and second terminations, in particular to derive information relating to at least one effect of the treatment.

Such a treatment can in particular be a cosmetic treatment. In particular, the treatment whose effect is to be evaluated may be a treatment intended to relieve or reduce the signs of skin aging or a disorder of the scalp. The biological activity and expression of a polypeptide can be determined as previously indicated. According to another aspect, the present invention relates to a method of cosmetic treatment of the signs of cutaneous aging, comprising at least one step of applying to at least part of the skin, mucous membranes and / or keratinous fibers, at least a cosmetic composition according to the invention. In another aspect, the present invention relates to the use of an effective amount of at least one polypeptide according to the invention or at least one agent modulating the expression of said polypeptide for the preparation and / or the improvement of a pluristratified cellular model, in particular of the epidermal or mucosal type, and in particular a reconstructed skin model. Within the meaning of the invention, the term "reconstructed skin model" is intended to denote a model in which various cell types are associated, such as, in particular, the natural constituents of the skin, such as, for example, keratinocytes, fibroblasts, Langerhans cells and melanocytes. The fibroblast type cells may be irradiated or not. Such models and their preparation are known to those skilled in the art.

Within the meaning of the present invention, one must be understood, unless otherwise indicated, in the sense of at least one. The examples given below are presented for illustrative and non-limiting purposes of the invention.

EXAMPLE I Analysis of samples taken by varnished stripping on different skin zones of an individual The analyzes are carried out using varnished strippings carried out on the legs.

Subjects suitable for study are grouped into 4 groups. Group AS corresponds to group 1: dry menopause n = 15. The AN group corresponds to group 2: normal menopause n = 13. Group JS corresponds to group 3: young dry n = 16. Group JN corresponds to group 4: normal young n = 14. 1: Preparation of acetone powders Two varnished strippings (B. Mehul et al., J Biol Chem 2000, Apr. 28; 275 (17): 12841-7) of 10 cm are arranged in 20 ml of acetone. The corneocytes take off. The mixture is filtered through a 40 m nylon membrane. Three successive rinses are performed with the same volume of acetone. The suspension is finally filtered on a vacuum pump. Acetonitrile powders of stratum corneum are obtained in dry form. 2: Extraction of the samples Extraction is carried out under denaturing conditions. To do this, prewashing is carried out with a volume (100 l) of buffer PBS (Phosphate Buffer Saline) + 0.1% Triton X100 which is added per mg of acetone powders. The mixture is ground with the potter and centrifuged. The corneocyte pellet is collected. It is extracted with the same volume (100 l / mg) of Laemmli buffer 0.0625 mM tris pH 6.8, 200 mM DTT, 2% SDS, 10% glycerol. The mixture is boiled for 10 minutes, then it is ground and centrifuged for 10 minutes at 10,000 g. The supernatant is collected. A protein assay is performed according to the Bradford technique with the Bradford reagent (Bio-Rad Protein assay). The samples are adjusted to 1 mg / ml. 3: Protein analyzes by Western blot The proteins are separated by SDS-PAGE electrophoresis. After semi-dry membrane transfer of PVDF (Immobilon-P Milipore) according to a standard protocol, the proteins are incubated with the primary anti-transglutaminase 3 antibody (Covalab 17CE) overnight at 4 ° C. Then the second incubation takes place with the secondary anti-rabbit antibody IgG-HRP conjugate, Bio-Rad) directed against the primary antibody 1:30 at room temperature. The presence of transglutaminase 3 on the membrane is revealed by immunodetection using the ECL Plus kit (Amersham). The membrane is then stained with Amidoblack to detect the total proteins present on the membrane. The image is acquired with FluorSmax (Biorad) and quantified bands using Quantity-one software (Biorad).

4: Results The results are expressed as delta cnt * mm2 of the protein of interest / delta cnt * mm2 of the total proteins.

Methodology - 2-way analysis of variance Age and Skin type taking into account the interaction of these two factors + 1-factor analysis of variance (group) and test of 25 Tukey multiple comparisons. The conditions of normality and homoscedasticity not being verified, the analysis was carried out after logarithmic transformation. The statistical analysis was carried out with the software SAS version 8.2 and SPSS version 12. All the tests were carried out at the bilateral threshold of 5%.

The following table shows the averaged results and their standard deviations at the mean (wk). transglutaminase group 3 sem transglutaminase 3 AS 532 194 AN 690 185 JS 1070 116 JN 1266 299 There was a significant decrease in the expression of transglutaminase 3 during skin aging (p = 0.002).

Claims (18)

1. Cosmetic use of an effective amount of at least one amino acid sequence polypeptide encoded by a nucleic acid sequence represented in whole or in part by a sequence represented by SEQ ID NO 1, an analogue thereof , or a fragment thereof, of at least one nucleic sequence coding for such a polypeptide or at least one agent modulating the activity or expression of such a polypeptide as a useful agent for prevent and / or treat the signs of skin aging. 2. Use of an effective amount of at least one amino acid sequence polypeptide encoded by a nucleic acid sequence represented in whole or in part by a sequence represented by SEQ ID NO 1, an analogue thereof, or a fragment thereof, at least one nucleic sequence coding for such a polypeptide or at least one agent modulating the activity or expression of such a polypeptide for the preparation of a therapeutic composition intended to prevent and / or treat the signs of skin aging. Use according to claim 1 or 2, wherein said polypeptide has an amino acid sequence represented in whole or in part by a sequence represented by SEQ ID NO 2, an analogue thereof, or a fragment thereof . 4. Use according to one of claims 1 to 3 wherein said polypeptide has an amino acid sequence selected from 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, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. Use according to any one of the preceding claims, wherein the modulating agent is an activator of the expression of said polypeptide. 6. Use according to the preceding claim, wherein said activator is selected from agents capable of mobilizing intracellular calcium, transcription factors Spl and Ets. The use according to any one of claims 1 to 4, wherein the modulating agent is an enzymatic activator of said polypeptide. 8. Use according to the preceding claim, wherein said activator is selected from cathepsin D, thapsigargin, sphingosine phosphorylcholine, CLSP, or certain fragments of this protein and GTP analogs. 9. Use according to any one of the preceding claims, wherein said composition is intended to prevent and / or treat a thinning of an epidermis and / or a loss of firmness, elasticity, density, and / or tonicity of an epidermis and / or the formation of wrinkles and fine lines. 10. Use according to any one of claims 1 to 8, wherein said composition is intended to prevent and / or treat cutaneous signs of dryness, in particular to prevent and / or treat dehydration of an epidermis. 11. Use of at least one polypeptide as defined in one of claims 1 to 4, as a tool for screening biological or chemical compounds capable of modulating the expression and / or the biological activity of said polypeptide. . 12. Use of at least one polypeptide as defined in one of claims 1 to 4, or at least one nucleic acid sequence coding for said polypeptide as an in vitro or ex vivo characterization tool. a state of an epithelium. 13. Use according to the preceding claim, wherein the state of the epithelium is selected from the dryness of an epidermis, chronological aging and photoaging. A method of characterizing a state of an epithelium comprising at least the steps of: a) determining in a sample of said epithelium a content of a polypeptide as defined in any one of claims 1 to 4 or a nucleic acid sequence encoding said polypeptide, and b) comparing said content determined in step a) with a reference value. 15. Method according to the preceding claim, characterized in that it is non-invasive. 16. A method for screening anti-aging active agents comprising at least the steps of: a) contacting at least one cell type capable of expressing a polypeptide as defined according to one of claims 1 to 4 with at least one chemical or biological compound to be tested, under conditions conducive to a manifestation of the expression of said polypeptide, b) determining a content of said polypeptide. 17. Cosmetic process for characterizing the effectiveness of a cosmetic or therapeutic treatment intended to supplement the signs of cutaneous aging, comprising at least the qualitative or quantitative characterization of the expression and / or the biological activity of a polypeptide such as as defined in one of claims 1 to 4. 18. Use of an effective amount of at least one polypeptide as defined in any one of claims 1 to 4, or at least one agent modulating the expression of said polypeptide for the preparation and / or the improvement of a pluristratified cellular model, in particular a reconstructed skin model.