|Ph.D Student||Bercovich Beatrice|
|Subject||Involvement of Phosphorylation and the Heat Shock Cognate|
Protein Hsc70 in Mechanisms of Substrate
Recognition by the Ubiquitin Proteolytic
|Department||Department of Medicine||Supervisor||? 18? Aaron Ciechanover|
Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substate. Conjugation is catalyzed via a three-step mechanism that involves three enzymes: E1, E2, and E3. E1 activates ubiquitin and E2 catalyzes transfer of the activated moiety to the substrate that is bound to E3. The tagged protein is degraded by the 26S proteasome. Targeting of certain proteins requires association with ancillary proteins and/or post-translational modifications. This work describes two distinct mechanisms by which different substrates are recognized by the ubiquitin system: A. Identification of the E2 enzymes involved in the signal-induced degradation of phosphorylated IkBa, the inhibitor of the transcription factor NF-kB: since activation of NF-kB requires signal-induced, ubiquitin- and proteasome- mediated degradation of IkBa, we searched for the ubiquitin carrier proteins (E2’s) involved in the process. We have shown that the two highly homologous E2 enzymes, UbcH5b and UbcH5c, and Cdc34/Ubc3 are the E2 enzymes specifically involved in targeting of the phosphorylated inhibitor both in vitro and in vivo. These enzymes are not involved in targeting the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. B. Involvement of the molecular chaperone Hsc70 in the ubiquitin-mediated degradation of certain protein substrates in vitro: we have demonstrated that Hsc70 immunodepletion and removal of K+ from rabbit reticulocyte lysate inhibited the conjugation and degradation of actin, a-crystallin, glyceraldehyde-3-phosphate dehydrogenase, a-lactalbumin, and histon H2A, but not that of bovine serum albumin, and lysozyme. Reconstitution was possible by supplementing the reactions with Hsc70 and K+. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site.