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Biotech / Medical : Millennium Pharmaceuticals, Inc. (MLNM) -- Ignore unavailable to you. Want to Upgrade?

To: Icebrg who wrote (2784)	1/1/2007 2:39:52 PM
From: Icebrg	Read Replies (1) \| Respond to of 3044

Importance of the Different Proteolytic Sites of the Proteasome and the Efficacy of Inhibitors Varies with the Protein Substrate* Alexei F. Kisselev12, Alice Callard1, and Alfred L. Goldberg, A Senior Fellow of the Ellison Foundation3 From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 The relative importance of the different proteolytic sites in mammalian proteasomes in protein degradation has not been studied systematically. Nevertheless, it is widely assumed that inhibition of the chymotrypsin-like site, the primary target of the proteasome inhibitors used in research and cancer therapy, reflects the degree of inhibition of protein breakdown. Here we demonstrate that selective inactivation of the chymotrypsin-like site reduced degradation of model proteins by pure 26 S proteasomes by only 11-50% and decreased only slightly the breakdown of proteins in HeLa cells. Inactivation of the caspase-like site decreased breakdown of model proteins by 12-22% and of the trypsin-like site by 3-35%. The relative contributions of these different sites depended on the protein substrate, and the importance of the trypsin-like sites depended on the substrate's content of basic residues. Simultaneous inhibition of the chymotrypsin-like and the caspase- or trypsin-like sites was needed to reduce degradation by >50%. Thus, 1) all three types of active sites contribute significantly to protein breakdown, 2) their relative importance varies widely with the substrate, 3) assaying the chymotrypsin-like activity overestimates the actual reduction in protein degradation, and 4) inhibition of multiple sites is required to markedly decrease proteolysis.

To: Icebrg who wrote (2784)	6/5/2007 12:33:21 PM
From: tuck	Read Replies (2) \| Respond to of 3044

[Velcade and gossypol versus melanoma] >>Cell Death Differ. 2007 Jun 1; [Epub ahead of print] Therapeutic window for melanoma treatment provided by selective effects of the proteasome on Bcl-2 proteins. Wolter KG, Verhaegen M, Fernández Y, Nikolovska-Coleska Z, Riblett M, Martin de la Vega C, Wang S, Soengas MS. [1] 1Department of Dermatology, University of Michigan, Comprehensive Cancer Center, Ann Arbor, MI, USA [2] 2Department of Surgery, University of Michigan, Comprehensive Cancer Center, Ann Arbor, MI, USA. Melanoma cells depend on sustained proteasomal function for survival. However, bortezomib, the first proteasome inhibitor in clinical use, is not sufficient to improve the poor prognosis of metastatic melanoma patients. Since the proteasome is also expressed in all normal cell compartments, it is unclear how to enhance the efficacy of bortezomib without exacerbating secondary toxicities. Here, we present pharmacological and genetic analyses of mechanisms of resistance to proteasome inhibition. We focused on Bcl-2, Bcl-x(L) and Mcl-1 as main antiapoptotic factors associated with melanoma progression. Despite an efficient blockage of the proteasome, bortezomib could not counteract the intrinsically high levels of Bcl-2 and Bcl-x(L) in melanoma cells. Moreover, Mcl-1 was only downregulated at late time points after treatment. Based on these results, a combination treatment including (-)-gossypol, an inhibitor of Mcl-1/Bcl-2/Bcl-x(L), was designed and proven effective in vivo. Using a specific RNA interference approach, the survival of bortezomib-treated melanoma cells was found to rely primarily on Mcl-1, and to a lesser extent on Bcl-x(L) (but not on Bcl-2). Importantly, neither Mcl-1 nor Bcl-x(L) inactivation affected the viability of normal melanocytes. This hierarchical requirement of Bcl-2 family members for the maintenance of normal and malignant cells offers a therapeutic window to overcome melanoma chemoresistance in a tumor cell-selective manner.<< Unclear to me what is meant by (-)-gossypol. Does the double negative mean just gossypol, or what? I've seen this in genetic studies, where it means the gene in question was entirely (both alleles) knocked out, but I don't understand it in this context. Illumination appreciated. Cheers, Tuck