Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-28
2004-05-04
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S252300, C435S320100, C514S002600, C530S350000, C536S023100, C930S010000
Reexamination Certificate
active
06730486
ABSTRACT:
The present invention relates to a novel human protein which is involved in the targeting of proteins towards proteasome degradation pathways. This protein, called h-&bgr;TrCP, is capable of interacting notably with the Vpu protein of HIV-1 virus and with the cell proteins I&kgr;B, &bgr;-catenin and Skp1p.
The degradation of proteins by proteasome, a multiprotein complex present in all cells, is involved in numerous essential cell phenomena such as the control of cell proliferation, the renewal of proteins and the removal of incorrectly folded proteins, particularly in the endoplasmic reticulum (CIECHANOVER A., Cell, 79, 13-21,1994). Numerous viruses, like HIV-1 virus, which degrades CD4 via one of its proteins Vpu (MRONO D., Cell, 82, 189-1992, 1995), exploit these cell pathways of protein degradation, in which the proteins are targeted towards proteasome by various interactions with other proteins before being degraded. To be targeted towards and degraded by proteasome, the proteins must generally be ubiquitinylated beforehand by ubiquitin-ligase complexes. Furthermore, to be ubiquitinylated, the proteins must often undergo modifications such as phosphorylations (CIECHANOVER A., Embo. J., 17, 7151-7160, 1998).
Several other proteins of the &bgr;TrCP type are known at the present time.
the &bgr;TrCP protein of Xenope, described by Spevak et al. (Mol. Cell. Biol., 13, 4953-4966, 1993);
the Slimb protein of drosophila, described by Jiang et al. Nature, vol. 391, Jan. 29, 1998); and
the KIAA 0696 protein identified by Ishikawa et al. (DNA Research, 5, 169-176, 1998) during a systematic analysis of sequences expressed in the brain.
Jiang et al. showed that the Slimb protein of drosophila is involved in the stability of the Armadillo protein and the signaling of two metabolic pathways essential for development, namely the Hedgehog and Wingless pathways. They also showed that the Slimb protein has a homology of about 80% with the &bgr;TrCP protein of Xenope, none of whose functions was described by Spevak et al. As the &bgr;-catenin of Xenope or man, which is the homolog of the Armadillo protein of drosophila, seems to be targeted towards proteasome degradation pathways in the absence of signaling of the Hedgehog and Wingless pathways, said authors suggest that, in man, the genes coding for the homologs of Slimb could be involved in the proteolytic degradation of &bgr;-catenin, a protein which acquires oncogenic properties when it is not degraded (POLAKIS P., Biochim. Biophys. Acta, 1332, F127-47, 1997).
However, despite the fact that conservation of the Wingless and Hedgehog pathways in vertebrates is important, it is not certain that the functions of the homologous proteins will be totally conserved. Moreover, there are numerous examples which show that there are always significant differences between species.
Also, solely on the basis of genetic studies, Jiang et al,. established the involvement of Slimb in the control of the Wingless and Hedgehog pathways in drosophila. Proof that this control is dependent on a direct interaction between Slimb and Armadillo, for example, has neither been sought nor found.
The protein according to the invention, called h-&bgr;TrCP, is capable of interacting with virus proteins or cell proteins which can act as mediators or be degraded by proteasome. In particular, the h-&bgr;TrCP protein is capable of interacting notably with the Vpu protein of HIV-1 virus and with the cell proteins I&kgr;B and &bgr;-catenin.
It is particularly useful for screening therapeutic agents such as, in particular, antitumoral, antiviral, anti-inflammatory and anti-Alzheimer agents.
The Vpu protein is a small membrane protein of 81 amino acids which is expressed by the majority of isolates of HIV-1 virus but not by those of the considerably less pathogenic FUV-2 virus or by those of SIV simian virus (COHEN et al., Nature, 334, 532-534, 1988, and STREBEL et al., Science, 2, 1221-1223, 1988).
One of the functions of the Vpu protein is its capacity to induce degradation of the CD4 protein, a cell receptor of HIV-1 virus, so it participates in reducing the expression of the CD4 receptor on the cell surface (Willey et al., J. Virol., 68, 1207-1212, 1994).
It is also known that the two phosphorylation serines of the Vpu protein, located in positions 52 and 56, are essential for the degradation of CD4 induced by Vpu (MARGOTIN et al., Virology, 223, 381-386, 1996). Moreover, during the process of infection by HIV-1 in the absence of the Vpu protein, the Gp160 envelope precursor and the newly synthesized CD4 protein combine in the endoplasmic reticulum to block the maturation of the Gp160 protein (BOUR et al., J. Virol., 65, 6387-6396, 1991. Degradation of the CD4 receptor mediated by the Vpu protein is essential for releasing the viral envelope protein which is held in the endoplasmic reticulum by being bound to CD4 through interaction with the Gp120 subunit, and for allowing the normal maturation of the envelope into the plasmic membrane and subsequently its integration into the virus particles, rendering them infectious. Recent studies have demonstrated the fact that degradation of the CD4 receptor mediated by the Vpu protein is sensitive to specific proteasome inhibitors and is dependent on the presence of an “intact ubiquitinylation machinery” (FUJITA et al., J. Gen. Virol., 78, 619-625, 1997).
Thus the Vpu protein participates in absolutely critical functions for assuring the production of large numbers of infectious virus particles, since it acts not only on the products of the gag gene, i.e. on the structural proteins, to increase the release of the virus particles, but also on the products of the env gene to allow the maturation of the envelope protein following degradation of the CD4 receptor. In 1996, MARGOTTIN et al. (supra) showed that the interaction between Vpu and CD4 took place via their cytoplasmic domain and that this interaction was not sufficient to trigger degradation of the CD4 receptor.
The Skp1p protein is a cell protein involved in the targeting of proteins towards proteasome degradation pathways, which depends on the ubiquitinylation of the proteins (PICKART C. M., The Faseb Journal, 11, 1055-1066,1997).
BAI et al. (Cell, 86, 263274, 1996) showed that the Skp1p protein was necessary for ubiquitin-mediated proteolysis and that this degradation took place due to the interaction of Skp1p with proteins containing a unit called F-box.
The Skp1p protein is an essential factor in the targeting of cell cycle regulatory proteins by proteasome. Targeting of the degradation of these regulators is particularly necessary when the cell cycle enters the S phase of DNA synthesis (PAGANO M., The Faseb Journal, 11, 1068-1075, 1997). Recent studies showed that the Skp1p protein and F-box proteins are the essential elements of high-molecular complexes called SCF (Skp1p-Cullin-F-box-protein complexes). These SCF complexes play the role of enzyme E3; through their ubiquitin-ligase activity, they allow the last step of the ubiquitinylation of substrate proteins, which are thus targeted towards degradation by proteasome (HOYT A., Cell, 91, 149-151, 1997). It is further pointed out that no Skp1p homolog has yet been identified in drosophila.
The I&kgr;B protein, which exists in different forms (&agr;, &bgr;, &egr;), is the major inhibitor of the NF&kgr;B transcription factor, keeping it in the form of an inactive complex in the cytoplasm (Beg A. et al., Genes and Dev., 7, 2064-2070, 1993). After stimulation of the cells by factors such as interleukin-1 (IL1) and tumor necrosis factor (TNF), the I&kgr;B protein is phosphorylated on serine residues S32 and S36. This phosphorylation leads very rapidly to the ubiquitinylation of the protein and to the targeting thereof towards degradation by proteasome. The active NF&kgr;B factor, for example in the form of two subunits P50 and P65, is then released and imported into the nucleus, where it will be able to activate a very large number of genes and cause inflammatory phenomena in particular.
The &bgr;-catenin protein is a cell prote
Arenzana Seisdedos Fernando
Benarous Richard
Concordet Jean-Paul
Durand Herve
Kroll Mathias
Antonelli Terry Stout & Kraus LLP
Institut National de la Sante et de la Recherche Medicale
Low Christopher S. F.
Schnizer Holly
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