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1.
Mol Cancer Ther ; 8(8): 2286-95, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19671755

ABSTRACT

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.


Subject(s)
Indenes/pharmacology , Protease Inhibitors/pharmacology , Pyrazines/pharmacology , Tumor Suppressor Protein p53/metabolism , Ubiquitin Thiolesterase/antagonists & inhibitors , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Tumor Suppressor Protein p53/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Peptidase 7
2.
J Biol Chem ; 283(24): 16762-71, 2008 Jun 13.
Article in English | MEDLINE | ID: mdl-18378672

ABSTRACT

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.


Subject(s)
Carrier Proteins/physiology , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptor, Melatonin, MT1/metabolism , Amino Acid Motifs , Animals , Carrier Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Kinetics , Models, Biological , Pituitary Gland/metabolism , Protein Binding , Receptors, Melatonin/metabolism , Sheep , Signal Transduction , Two-Hybrid System Techniques , Valine/chemistry
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