Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-03-15
2001-01-30
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S252300, C536S023200
Reexamination Certificate
active
06180386
ABSTRACT:
The present invention relates to a DNA sequence coding for a new human protein Tx related to the interleukin-1beta converting enzyme, the protein Tx, their production process, the pharmaceutical compositions containing it and their uses as medicaments.
Interleukin-1beta (IL-1&bgr;) is a pro-inflammatory cytokine involved in the pathogenesis of multiple acute or chronic inflammatory illnesses such as rheumatoid arthritis, inflammatory illnesses of the intestines or the septic shock (Dinarello et al., 1992, Immunological Reviews, 127, 119-146).
The human monocytes and macrophages synthesize IL-1&bgr; in the form of an inactive precursor of 31 kDa (pIL-1&bgr;). The pIL-1&bgr; does not have the conventional signal sequence and can only be secreted efficiently by the cell after cleavage between the aspartic acid 116 and the alanine 117. This cleavage, which generates the active IL-1&bgr; form of 17 kDa, is carried out by a specific enzyme called interleukin-1beta converting enzyme (ICE) (Thornberry et al., 1992, Nature, 356, 768-774; Cerretti et al., 1992, Science, 256, 97-100). This enzyme has been characterized and cloned in man and in mice (Nett et al., 1992, Journal of Immunology, 149, 3254-3259; Molineaux et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 1809-1813). It is a unique cysteine protease which has no homology with other known thiol-proteases. It also possesses a particular specificity for certain Asp-X peptide bonds of pIL-1&bgr;.
The ICE enzyme is composed of two sub-units of 20 kDa (p20) and 10 kDa (p10) whose combination is necessary for enzymatic activity. These sub-units originate from the proteolytic cleavage of a pro-enzyme form of 45 kDa (p45). The ICE enzyme itself is capable of cleaving its precursor p45 or the p30 form of 30 kDa, which does not have the 119 amino acids of the N-terminal part of the pro-enzyme, in the p20 plus the p10 active form. The complete sequence of p45 has been characterized by its cDNA as well as the amino acid sequence (Thornberry et al. already quoted). The characterization of the gene of human ICE has been described (Cerretti et al., 1994, Genomics, 20, 468-473).
Recent work has revealed a possible role of ICE in the regulation of programmed cellular death or apoptosis (Yuan et al., 1993, Cell, 75, 641-652). In fact, ICE has a 28% homology with Ced-3, a protein of C. elegans involved in apoptosis and the superexpression of murine ICE in rat fibroblasts which triggers apoptosis (Miura et al., 1993, Cell, 75, 653-660). Moreover, the expression of the protein crmA, an inhibiting viral serpine of ICE, in ganglioneurons of transfected chickens protects these cells from death by apoptosis induced by the suppression of growth factor (nerve growth factor) (Gagliardini et al., 1994, Science, 263, 826-828). These observations suggest that ICE or homologues of this protein could be involved in the regulation of programmed cellular death observed in particular in degenerative neuronal illnesses such as Alzheimer's disease or Parkinson's disease as well as in cerebral ischemia (Barinaga, M., Science, 259, 762, 1993).
The revelation of new proteins related to ICE playing a role either in the maturation of IL-1&bgr; or in apoptosis may contribute to the development of new therapeutic or diagnostic agents in situations in which IL-1&bgr; or apoptosis are involved.
The present invention relates to a new human protein Tx which has an approximately 52% homology with the human precursor p45 of ICE and which does not allow the maturation of the precursor of IL-1&bgr; into active cytokine. The protein Tx has two unexpected functions: on the one hand, it is a protease and it is in particular capable of cleaving the precursor p30 of ICE into sub-units p10 and p20 and on the other hand, it is capable of inducing apoptosis in cells, for example in transfected Cos cells.
These biological properties allow the use of the protein Tx to be anticipated in the treatment of pathological situations which respond to IL-1&bgr; or in which apoptosis occurs.
The present invention also relates to a new human protein Ty which has a homology of greater than 70% with the protein Tx. The protein Ty is capable of inducing apoptosis in cells, for example, in transfected Cos cells. The protein Ty is a protease which is capable of self-cleaving in an intermolecular manner.
Therefore a subject of the present invention is a DNA sequence containing a DNA sequence coding for a human polypeptide having a protease activity and having the nucleotide sequence of the sequence SEQ ID N
o
1:
GCTCTTTCCA ACGCTGTAAA AAAGGACAGA GGCTGTTCCC T ATG GCA GAA GGC
53
Met Ala Glu Gly
1
AAC CAC AGA AAA AAG CCA CTT AAG GTG TTG GAA TCC CTG GGC AAA GAT
101
Asn His Arg Lys Lys Pro Leu Lys Val Leu Glu Ser Leu Gly Lya Asp
5 10 15 20
TTC CTC ACT GGT GTT TTG GAT AAC TTG GTG GAA CAA AAT GTA CTG AAC
149
Phe Leu Thr Gly Val Leu Asp Asn Leu Val Glu Gln Asn Val Leu Asn
25 35
TGG AAG GAA GAG GAA AAA AAG AAA TAT TAC GAT GCT AAA ACT GAA GAC
197
Trp Lys Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala Lys Thr Glu Asp
40 45 50
AAA GTT CGG GTC ATG GCA GAC TCT ATG CAA GAG AAG CAA CGT ATG GCA
245
Lys Val Arg Val Met Ala Asp Ser Met Gln Glu Lys Gln Arg Met Ala
55 60 65
GGA CAA ATG CTT CTT CAA ACC TTT TTT AAC ATA GAC CAA ATA TCC CCC
293
Gly Gln Met Leu Leu Gln Thr Phe Phe Asn Ile Asp Gln Ile Ser Pro
70 75 80
AAT AAA AAA GCT CAT CCG AAT ATG GAG GCT GGA CCA CCT CAC TCA GGA
241
Asn Lys Lya Ala His Pro Asn Met Glu Ala Gly Pro Pro Glu Ser Gly
85 90 95 100
GAA TCT ACA GAT GCC CTC AAG CTT TGT CCT CAT GAA GAA TTC CTG AGA
389
Glu Ser Thr Asp Ala Leu Lys Leu Cys Pro His Glu Glu Phe Leu Arg
&
Diu Anita
Faucheu Chi
Hercend Thierry
Lalanne Jean Louis
Livingston David J
Achutamurthy Ponnathapu
Bierman, Muserlian and Lucas
Hoechst Marion Roussel
Moore William W.
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