Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2001-07-19
2003-11-18
Monshipouri, Maryam (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S320100, C435S325000, C435S252300, C435S006120, C536S023200
Reexamination Certificate
active
06649388
ABSTRACT:
The present invention relates to the field of biology and of the regulation of the signal transduction pathways which respond to extracellular stimuli. More specifically, the present invention is concerned with novel polypeptides which are derived from the human JNK3 protein, their variants, the corresponding nucleotide sequences, and their uses.
The protein JNK (c-jun N-terminal kinase), also termed SAPK (stress-activated protein kinase), belongs to the family of MAP (mitogen-activated protein) kinases. It is involved in the signal transduction pathways which respond to extracellular stimuli (for example, proinflammatory cytokines or environmental stresses). Its activation requires the phosphorylation of threonine 221 and tyrosine 223 within a highly conserved T-P-Y tripeptide motif located in the kinase domain (Dérijard et al. 1994). The substrates of JNK are transcription factors such as c-jun (phosphorylated on serine 63 and serine 73), ATF-2 (phosphorylated on threonine 69 and threonine 71) and Elk-1.
The JNK proteins exhibit a large number of isoforms, and ten isoforms have been recorded to date. They derive from three different genes, termed JNK1, JNK2 and JNK3. The JNK1 and JNK2 genes encode two isoforms, &agr; and &bgr;, by means of alternative splicing (Gupta et al. 1996). For each isoform, &agr; and &bgr;, there exists a short version and a version which is elongated C-terminally. The short version is linked to the insertion, during the alternative splicing, of five nucleotides which supply a termination codon.
The proteins JNK3&agr;1 and JNK3&agr;2 are the only two isoforms of JNK3 which have so far been recorded. They differ from JNK1 or JNK2 by, inter alia, an N-terminal extension of 38 amino acids whose function has not yet been established. The rat JNK3 homologue, termed SAPK&bgr;, does not exhibit this distinctive feature.
The tissue distribution of the isoforms is very diverse, with variable levels of expression. However, it has been demonstrated that, while the JNK3 isoforms are more selectively expressed in the brain (for example in the hippocampus or in the cerebellum (Mohit et al. 1995)), they are also expressed in the heart and the testes.
While a growing number of results underline the importance of JNK3 in the phenomena of neurodegeneration and neuronal death by apoptosis, the mode of action of JNK3 remains unknown.
It has been shown that the neurones of the CA1 region of the hippocampus of patients who have suffered a period of hypoxia exhibit strong JNK3 immunoreactivity within the nuclei whereas JNK3 immunoreactivity is diffuse and exclusively cytoplasmic in control samples (Zhang et al. 1998). Furthermore, deletion of the JNK3 gene in mice results in resistance to kainic acid, which is an agonist of the glutamate receptors involved in the phenomena of excitotoxicity. The authors (Yang et al. 1997) provide a detailed description of the reduction in the epileptic effects and the prevention of the neuronal death by apoptosis in the hippocampus following injection of kainic acid into these mice which have been deleted for JNK3. Lastly, one of the preferred substrates of JNK3 is the transcription factor c-Jun, which is one of the components of the AP1 complex, which is itself heavily involved in functions of survival and neuronal degeneration. The c-Jun factor appears to have a double function i.e. both in cell death and in neuronal protection (Herdegen et al. 1997). Suppression of the expression of c-Jun or inhibition of its function protects the hippocampal and sympathetic neurones from cell death in culture (Estus et al. 1994, Ham et al. 1995). Finally, while expression of c-Jun is increased in apoptotic neurones which are degenerating following ischaemia, nerve section or irradiation, it is also increased in biopsies taken from patients afflicted with neurodegenerative pathologies such as multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer's disease, Parkinson's disease and Huntington's disease (Anderson et al. 1994, Herdegen et al. 1998, Martin et al. 1996).
While the JNK3 protein kinase nowadays appears to be one of the key elements involved in neuronal degeneration, the precise nature of its mode of action is unknown. In this regard, the identification of new natural isoforms of JNK3 represents a major challenge for understanding the mechanism of action of this protein in the phenomena of neuronal degeneration and for identifying novel targets for aiming at therapeutically.
The present invention describes the detection, cloning and characterization of novel polypeptides derived from JNK3.
The present invention results from characterizing three novel isoforms of human JNK3 termed hJNK3&agr;139, JNK3&Dgr;N&agr;1 and JNK3&Dgr;N&agr;2. It ensues, more particularly, from the demonstration that two of these isoforms, i.e. JNK3&Dgr;N&agr;1 and JNK3&Dgr;N&agr;2, lack the N-terminal extension which characterizes the known isoforms of JNK3 and the demonstration that these isoforms JNK3&Dgr;N&agr;1 and JNK3&Dgr;N&agr;2 exhibit properties which are different from those of the previously described isoforms of JNK3. It furthermore ensues from the discovery that these novel isoforms which lack the N-terminal extension unexpectedly share properties in common with the JNK1&bgr;1 and JNK2&agr;1 isoforms.
The identification of these novel isoforms of JNK3 makes it possible to envisage a large number of applications. These applications cover, in particular, identifying novel neuroprotective compounds which are able to interact specifically with these isoforms. These compounds can be used for preventing and treating different pathologies which are brought about by neuronal degeneration, among which may be mentioned Alzheimer's disease, Huntington's disease and Parkinson's disease, senile dementia and dementia due to AIDS, cranial traumas, cerebral oedemas, hypoxias and anoxias. These novel isoforms can also be used in molecular modelling for achieving an improved understanding of the structure and function of these enzymes and their involvement in pathologies which implicate one or more isoforms of JNK3. Finally, these isoforms of JNK3 are also useful for detecting novel proteins which are involved in intracellular signalling pathways which are specific to each of them. It is thus possible to identify novel relevant targets which are involved in degenerative neuropathologies.
The invention firstly relates to polypeptides which are derived from the JNK3&agr;1 or JNK3&agr;2 isoforms and which contain an N-terminal or C-terminal deletion. According to a first embodiment, the derivatives are derivatives which contain an N-terminal deletion corresponding to the first 38 amino acids of these isoforms. According to another variant, the derivatives are derivatives which contain a deletion of the C-terminal amino acids from amino acid 139 onwards.
Preferably, the derivatives according to the invention are polypeptides which are selected from the sequences SEQ ID No. 23, SEQ ID No. 25 and SEQ ID No. 27, or a variant of these sequences.
Within the meaning of the invention, the term variant refers to any polypeptide whose structure differs from a polypeptide selected from the sequences SEQ ID No. 23 or SEQ ID No. 25 or SEQ ID No. 27 by one or more modifications of a genetic, biochemical and/or chemical nature and which preserves at least one of the biological properties of said polypeptide. The modification can, in particular, be any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives can be generated for different purposes, such as, in particular, that of improving their level of production, that of increasing their resistance to proteases or of improving their passage across cell membranes, that of increasing their therapeutic efficacy or of reducing their side effects, that of increasing the affinity of the polypeptides for their sites of interaction, or that of conferring novel pharmacokinetic and/or biological properties on it. Advantageously, the variants comprise deletions or m
Desanlis-Cremond Francine
Fournier Alain
Maury Isabelle
Zhou-Liu Qing
Aventis Pharma S.A.
Coppola William C.
Monshipouri Maryam
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