Isolated peptide of the horny layer and user thereof

Details Isolated peptide of the horny layer and user thereof Isolated peptide of the horny layer and user thereof

2002-07-24

2004-10-05

Wax, Robert A. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000, C514S863000, C514S078000, C536S063000, C424S134100

Reexamination Certificate

active

06800609

ABSTRACT:

The invention has as its object an isolated polypeptide from the family of the calcium-fixing proteins, a mixture of polypeptides obtained from the proteolysis of the isolated polypeptide, compositions that contain them, the uses of said polypeptide and a cosmetic treatment process that is intended to treat dry skin, hyperkeratosis, parakeratosis, psoriasis, ichthyosis, and neoplasias. The invention also has as its object a deoxyribonucleic acid sequence that codes for said polypeptide and the uses of said deoxyribonucleic acid sequence.
The key role of the calcium flows in the cellular functions has been extensively described for more than 20 years. The calcium is thus an intracellular messenger of outstanding importance. Numerous hormones and extracellular messengers exert their effect by an increase of the intracellular ratio of calcium. It was quickly shown that most of the effects of the calcium were determined by a large, relatively homogeneous family of calcium-binding proteins. Among this family, the calmodulins are the most ubiquitous. The role of the calmodulin is to recognize the changes in the concentration of cytosolic calcium ions and to transmit the information to the intracellular proteins.
The transmission of the signal is carried out when the calmodulin binds the calcium during an increase of the cell ratio of the latter. This brings about changes in conformation of the protein which increases considerably its affinity for the target protein.
In terms of configuration, the free calcium calmodulin contains two globular domains that are connected by a flexible arm. Each domain contains two well-defined “helix-loop-helix” patterns that are known under the name of “EF-Hands” and that are responsible for the bond with calcium. The bond with the calcium induces a very significant conformational change. This conformational change is reflected by a presentation of the globular hydrophobic portion of the protein that allows the calmodulin to recognize and to bind to certain proteins with a high affinity. The bond of the calmodulin with its target protein then leads to the creation of an active complex.
The calmodulin is known for interacting with numerous proteins or enzymes as well as with numerous hydrophobic pharmaceutical agents or else with peptides of natural origin or synthetic origin.
In this regard, it is possible to cite
Certain protein-kinases involved in the cellular homeostasis and in certain critical cellular functions such as metabolism, motility, the transcription of genes and the cellular division, the proliferation;
The calcineurin that is a phosphatase that has a role in the regulation of enzymes and proteins that are involved in the transduction of the calcium signal;
The phosphodiesterase with cyclic nucleotides, the guanidylcyclase and the adenylylcyclase thus regulating in a positive manner and/or in a negative manner the ratios of the AMP-cyclic and GMP-cyclic second messengers in the mechanism of action of certain hormones;
The three isoforms of the NO-synthase that occur in the production of NO and thus in the vascular relaxation, the cytotoxic action of the macrophages, the release of neurotransmitters, neuropeptides and/or hormones;
Certain vital cytoskeleton proteins in the monitoring of the shape of cells, their rigidity and their adhesion, such as, for example, myosin;
Caldesmon, spectrins, adducin, proteins that bind to actin;
Desmocalmin, a protein of the desmosome that interacts with the keratins;
The plasma-membrane Ca
2+
-ATPases that occur in the maintenance of the intracellular ion concentration;
Ornithine decarboxylase, certain A2 phospholipases and certain transglutaminases have been cited as being calmodulin-dependent.
In contrast, certain studies suggest that calmodulin has a role in the phenomena for reparation of deoxyribonucleic acid and that it potentially would be involved in aging of the skin.
It is known, finally, that if calmodulin is a well-described protein, it is actually the prototype of a large family of protein of which all of the individuals have not yet been described.
Thus, after long and laborious works, the applicant demonstrated, among the proteins of the epidermis, isolated and purified by biochemical techniques, a polypeptide that is expressed in the corneal epithelia. This polypeptide, otherwise called “CLSP” in the text (for skin protein similar to calmodulin: calmodulin-like skin protein) is expressed in the epidermis. It is known that in the corneal epithelia, the very large majority of expressed proteins is constituted by keratins. Thus, it is only by working out a particular extraction method that eliminates the keratins that the applicant was able to isolate and then to purify the CLSP.
The applicant then determined the primary amino acid sequence.
The invention therefore has as its object an isolated and purified polypeptide that belongs to the family of the calcium-fixing proteins (CaBP: calcium binding protein), characterized by the fact that it corresponds to the amino acid sequence SEQ ID NO: 1 below:
Met Ala Gly Glu Leu Thr Pro Glu Glu Glu
10

Ala Gln Tyr Lys Lys Ala Phe Ser Ala Val
20

Asp Thr Asp Gly Asn Gly Thr Ile Asn Ala
30

Gln Glu Leu Gly Ala Ala Leu Lys Ala Thr
40

Gly Lys Asn Leu Ser Glu Ala Gln Leu Arg
50

Lys Leu Ile Ser Glu Val Asp Ser Asp Gly
60

Asp Gly Glu Ile Ser Phe Gln Glu Phe Leu
70

Thr Ala Ala Arg Lys Ala Arg Ala Gly Leu
80

Glu Asp Leu Gln Val Ala Phe Arg Ala Phe
90

Asp Gln Asp Gly Asp Gly His Ile Thr Val
100

Asp Glu Leu Arg Arg Ala Met Ala Gly Leu
110

Gly Gln Pro Leu Pro Gln Glu Glu Leu Asp
120

Ala Met Ile Arg Glu Ala Asp Val Asp Gln
130

Asp Gly Arg Val Asn Tyr Glu Glu Phe Ala
140

Arg Met Leu Ala Gln Glu
146
The polypeptide of the invention can be of natural or synthetic origin. Synthetic is defined here as any polypeptide that is obtained chemically or by production in an organism after introduction into this organism of elements that are necessary for this production.
The polypeptide of the invention can be obtained from any possible origin, namely either animal, in particular mammals and even more particularly human, or vegetable, either microorganisms (virus, phages, bacteria, i.a.) or else mushrooms, without prejudging whether or not they are present in a natural manner in said organism of origin.
Preferably, the polypeptide of the invention is of natural origin, purified from mammal tissues, particularly from mammal skin.
Preferably, the polypeptide of the invention is purified from human skin and even more preferably from human epidermis.
It is known that in a polypeptide, one or more amino acid residues can change for amino acid residues that have a similar hydropathic index without thereby changing the biological properties of the polypeptide. The hydropathic index is an index that is attributed to amino acids based on their hydrophobicity and their charge (Kyte et al. (1982), J. Mol. Biol, 157: 105).
The invention thus also has as its object a polypeptide as described above in which at least one amino acid residue has been changed for an amino acid residue that has a similar hydropathic index.
It is known that in general, the mature polypeptides that are found in the cells are obtained from the maturation of precursors that contain in their sequence the sequence of the mature polypeptide.
The invention thus also relates to any polypeptide, natural or synthetic, whose sequence partly consists of the sequence of the polypeptide of the invention.
It is known that the polypeptides can undergo post-translational modifications such as the formation of disulfide bonds, specific proteolytic cleavages, the addition of glucides (glycosylation), phosphorylation in particular at the level of serines and/or threonines and/or tyrosines, and/or the combination with lipids.
The polypeptide of the invention may have undergone one or more post-translational modifications.
The invention thus also relates to the polypeptide of the invention that may or may not have undergone post-translational modifications.
It is known to classify the polypeptides based

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