Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-04
2003-06-24
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S325000, C530S350000, C536S023500
Reexamination Certificate
active
06582933
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a protein that inhibits activation of transcription factor NF&kgr;B.
More detailed, the present invention relates to p65-binding protein (called ReIA-associate inhibitor or abbreviated as RAI hereinafter) that binds to p65, which is a subunit of transcription factor NF&kgr;B, and that inhibits transcriptional activity of NF&kgr;B, a process for producing it, a cDNA encoding it, a vector comprising the cDNA, a host cell transformed with the vector, an antibody against the inhibitor, and a pharmaceutical composition comprising the inhibitor or the antibody.
Further, the present invention relates to recombinant production of these proteins (especially, in vivo production), a nucleotide encoding them, a vector for expression and duplication and treatment and/or prevention of adult respiratory distress syndrome (ARDS), asthma, Allograft rejection, inflammatory diseases (inflammatory arthritis, angitis etc.), ischemic diseases, autoimmune diseases including chronic rheumatism, metastasis and invasion of cancer, vasoreconstriction and diseases induced by NF&kgr;B besides above.
TECHNICAL BACKGROUND
An acceleration of expressing many and various genes is observed in inflammation. Such genes include ones encoding interleukin, transcription factors, cohesive molecules, and factors in coagulation system etc. NF&kgr;B which is a transcription factor has been said to relate to the transcription of such genes mostly.
It has been known that transcription factor NF&kgr;B is expressed in cytoplasm. The transcription and induction of gene due to NF&kgr;B-like proteins may be caused by activation of the protein. By such an activation, it becomes possible to translocate the transcription factor prepared in advance from cytoplasm to nucleus.
It is known that this translocation is controlled by phosphorylation and degradation of suppressor protein which is called for I&kgr;B.
Transcription factor NF&kgr;B was isolated from mature B cells in the form of binding to 10 nucleotide sequence motif in &kgr; light chain enhancer for the first time. Therefore, NF&kgr;B was thought to be specific for the generating stage of matured B cells. However, NF&kgr;B-like proteins have been identified in a lot of cells, so it is shown that such a factor relates to induction of gene transcription generally. Such fact has been confirmed by functional identification of an active type NF&kgr;B-binding position in some inducing genes.
NF&kgr;B is a heterodimer consisting of a subunit of 50 kDa (p50) and a subunit of 65 kDa (p65).
NF kappa B (NF&kgr;B), which is a nuclear factor, is a sequence-specific DNA-binding protein complex which regulates the expression of viral genomes, including the human immunodeficiency virus (HIV), and a variety of cellular genes, particularly those involved in immune and inflammatory responses. The members of the NF&kgr;B family in mammalian cells include the proto-oncogene c-Rel, p50/p105 (NF&kgr;B1), p65 (RelA), p52/p100 (NF&kgr;B2), and RelB etc.
All of these proteins share a conserved 300 amino acid region of homology known as the Rel homology domain (RHD), which is responsible for DNA binding, dimerization, and nuclear translocation of NF&kgr;B.
In most cells, Rel family members can form hetero- and homodimers with distinct specificities in various combinations. A common feature of the regulation of transcription factors belonging to Rel family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as IKBs. Treatment of cells with a variety of inducers such as phorbol esters, interleukin 1, tumor necrosis factor-&agr; (TNF-&agr;), viral infection and many mitogens and cytokines etc. results in the dissociation of the cytoplasmic complexes and translocation of free NF&kgr;B into the nucleus. The dissociation of the cytoplasmic complexes is known to be triggered by the phosphorylation and subsequent degradation of the IKB proteins. Such a degradation exposes the nuclear localization sequence in the remaining NF&kgr;B heterodimer, leading to nuclear translocation and subsequent binding of NF&kgr;B to DNA regulatory elements within NF&kgr;B target genes. The p65 subunit is frequently detected in NF&kgr;B complexes and has a strong transcription activation potential. p65 dimerizes with other NF&kgr;B family members and activates gene expression via its potent transactivation domain.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide a novel protein that inhibits transcriptional activity of NF&kgr;B (NF&kgr;B inhibitor), a process for producing it, a cDNA encoding it, a vector comprising the cDNA, a host cell transformed with the vector, an antibody against the inhibitor, and a pharmaceutical composition comprising the inhibitor or the antibody.
The present inventor et al. have thought that novel inhibitors may exist besides I&kgr;B which was known as an NF&kgr;B inhibitor. From the results of focusing their attention on and an extensive studies of NF&kgr;B, especially p65 subunit, the present inventor et al. have achieved to find out a novel NF&kgr;B inhibitor, confirmed amino acid sequence, a cDNA sequence encoding it, function of the inhibitor and tissue-distribution etc. and then competed the present invention.
RelA-associated inhibitor of the present invention is regarded as nucleus factor and inhibits NF&kgr;B-dependent transcription activation by inhibiting DNA-binding activity of NF&kgr;B. The gene encodes RelA-associated inhibitor is a gene having a high homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an SH3 domain.
RAI appeared to locate in nucleus with the same location pattern of TNF-&agr;-induced p65. In addition, RAI inhibits activation of NF&kgr;B, but does not show any influence on p53-dependent transcription activation in the cells which is transformed temporarily. Therefore, RAI is a novel p65-binding protein which relates to another mechanism of NF&kgr;B-dependent transcription regulation. The present inventor has confirmed these interaction in vitro with bacterially expressed fusion proteins and in vivo using immunoprecipitation/ Western blot assay.
In spite of its similarity to 53BP2, RAI did not shown any interaction with p53 in a yeast two-hybrid assay. The cDNA encodes this protein has a high structual homology to 200 amino acid at the C-terminal region of 53BP2 containing four ankyrin repeats and an SH3 domain that are important for protein-protein interactions.
In human, mRNA of RAI was specifically expressed in heart, placenta and prostate while it was markedly reduced in liver, skeletal muscle and peripheral blood leukocyte.
Recent rapid developments in techniques for constructing cDNAs and sequencing techniques have made it possible to quickly sequence a large amount of cDNAs. By utilizing these techniques, a process, which comprises constructing cDNAs library from various cells or tussles, cloning cDNAs at random, identifying the nucleotide sequences thereof, expressing novel polypeptides encoded by them to analyze its physiological function, is now in progress. Method of yeast two-hybridization has been known as one of such techniques.
By this technique, it becomes possible to identify a large amount of homologous proteins at the same time and to obtain the aimed protein only easily by inserting reporter genes such as fluorescent marker etc.
The present inventor has carried out experiments of such a yeast two-hybrid screen using NF&kgr;B protein subunit p65 as a probe to find a novel factor that binds to a novel p65 and that inhibits an activity of p65 and then found out the factor of the present invention.
The sequence of RelA-associated inhibitor of the present invention has been confirmed to be novel according to a homology search. Only a sequence of RelA-associated inhibitor of the present invention is a novel one among some clones obtained by the process for the sequence of the present invention described hereinafter.
The amino acid sequence of RelA-associated inhibitor of the present inventi
Chan Christina
Haddad Maher
Ono Pharmaceutical Co. Ltd.
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