intro_s13

intro_s13 - Introduction Session 13 PROTACS: CHIMERIC...

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Introduction Session 13 PROTACS: CHIMERIC MOLECULES TO TARGET PROTEINS FOR UBIQUITINATION AND DEGRADATION (taken from KM Sakamoto, Mol. Genetics and Metabolism 77 (2002) 44-56 ) To circumvent the problem of transducing cells at high efficiency, we sought to target deliberately a protein to the SCF complex by developing a chimeric compound, known as proteolysis targeting chimeric molecule (PTCM) . We first tested whether the PTCM could recruit methionine aminopeptidase-2 (MetAP-2) to the SCF β -TRCP for ubiquitination and degradation in vitro. A PTCM was synthesized that contained at one end the minimal 10 aa phosphopeptide sequence of I κ B that is recognized by the F-box protein β -TRCP and at the other end, the MetAP-2 binding compound, ovalicin. MetAP-2 binds to ovalicin. MetAP-2 binds to ovalicin covalently. We performed ubiquitination experiments with lysates from 293T cells transfected with Flag tagged β -TRCP and Flag tagged CUL1. The SCF complex was immunoprecipitated using Flag affinity beads followed by addition of purified E1, E2, ATP, and ubiquitin. Our results demonstrate that the PTCM could recruit the MetAP-2 to the SCF β -TRCP complex resulting in ubiquitination. Addition of PTCM also resulted in degradation of MetAP-2 in Xenopus extracts. To determine whether PTCM could be generalized to other ubiquitin ligases, we performed ubiquitination assays with Cbl. Cbl is a monomeric ubiquitin ligase that attaches ubiquitin to signaling molecules and receptor tyrosine kinases resulting in proteolysis. We generated a PTCM that consisted of ovalicin and the Zap70 phosphopeptide, which binds Cbl. Ubiquitination reactions were performed with purified Cbl, various E1s, Ubch4 (E2), ubiquitin, ATP, MetAP-2, and the Zap70-ovalicin PTCM. We demonstrated that PTCM promotes ubiquitination of MetAP-2 by Cbl in vitro. These results suggest that PTCM can be generalized to other ubiquitin ligases. Future work will focus on testing other targets that promote tumorigenesis, e.g., androgen receptor in prostate cancer cells. If cell permeable PTCMs prove to increase turnover and degrade proteins in cells, this would lead to potential therapeutic applications in patients with cancer and other diseases. Figure by MIT OCW. After Sakamoto, KM. Ubiquitin-dependent proteolysis: its role in the human diseases and the design of therapeutic strategies." Mol Genet Metab 77 1-2 (2002) 44-56. General application of PTCMs. A schematic representation of how different disease-promoting proteins might be recruited to E3 ligases for ubiquitination and degradation by specific PTCMs. Ub IIgase-1 General application of PTCMs. Ub IIgase-2 target 1 target 2 target 3 target 4 target 1 target 2 target 3 target 4
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BORTEZOMIB (also PS-341 or Velcade): A novel, first-in-class proteasome inhibitor for the treatment of multiple myeloma and other cancers (reviewed by P.G. Richardson et al. Cancer con rol (2003) 10: 361-369) t (A full text PDF of this article is available at http://www.moffitt.usf.edu/pubs/ccj/v10n5/pdf/361.pdf)
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This note was uploaded on 11/11/2011 for the course BIO 7.344 taught by Professor Bobsauer during the Spring '08 term at MIT.

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intro_s13 - Introduction Session 13 PROTACS: CHIMERIC...

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