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 substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, ␣-crystallin, glyceraldehyde-3-phosphate dehydrogenase, ␣-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K ؉ , which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.Degradation of short-lived and key regulatory proteins via the ubiquitin-proteasome pathway plays important roles in basic cellular processes. Protein targets of the ubiquitin system include, among others, cyclins, cyclin-dependent kinases and their inhibitors, tumor suppressors, oncoproteins, and transcriptional activators and their inhibitors. Selection of proteins for degradation can be mediated via primary (constitutive) or secondary signals such as post-translational modifications or via association with ancillary proteins. These signals are recognized by specific ubiquitin-protein ligases (E3), 1 to which the substrate proteins bind prior to ubiquitination. Thus, the ligases play a key role in the ubiquitin proteolytic cascade, recognition and selection of proteins for conjugation and subsequent degradation. Following formation of the polyubiquitin adduct, the protein moiety is degraded by the 26 S proteasome complex, and free and reutilizable ubiquitin is released (reviewed in Refs. 1-4).Molecular chaperones comprise a set of universally conserved proteins that bind and stabilize conformers of other proteins. A regulated, ATP-dependent association-dissociation cyc...
A new substrate for ubiquitin carboxyl-terminal hydrolase, the carboxyl-terminal ethyl ester of ubiquitin, has been synthesized by a trypsin-catalyzed transpeptidation. In the presence of 1.6 M glycylglycine ethyl ester, trypsin removes the carboxyl-terminal glycylglycine of ubiquitin and replaces it with the dipeptide ester. The equilibrium mixture under these conditions contains 30% ubiquitin ethyl ester and 70% hydrolysis product, the 74-residue fragment of ubiquitin. Ubiquitin ethyl ester can be purified by gel filtration and ion-exchange chromatography. The structure of this product has been verified by identification of the products of base hydrolysis, tryptic cleavage in aqueous solution, and peptide mapping. When ubiquitin ethyl ester is incubated with purified ubiquitin carboxyl-terminal hydrolase, specific cleavage of the ester linkage is observed. A rapid, sensitive assay is described utilizing high-performance liquid chromatography. By use of this assay, it has been shown that ubiquitin carboxyl-terminal hydrolase is inactivated in the absence of thiols. Optimal protective effects are seen with 10 mM dithiothreitol. The rate of catalysis is maximal at pH 8.5, with evidence for catalytically important groups with pK values of 5.2, 7.6, and 9.5. These findings are consistent with the participation of a thiol group in the active site. Native ubiquitin is a competitive inhibitor of ubiquitin ethyl ester hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
A free NH2-terminal group has been previously shown to be an obligatory signal for recognition and subsequent degradation of proteins in a partially fractionated and reconstituted ubiquitin proteolytic system. Naturally occurring proteins with acetylated NH2-termini--most cellular proteins fall in this category--were not degraded by this system. Other studies have suggested that the identity of the NH2-terminal residue is important in determining the metabolic stability of a protein in vivo (N-end rule). Whole reticulocyte lysate and antibodies directed against the ubiquitin-activating enzyme (E1) have now been used to show that such acetylated proteins are degraded in a ubiquitin-dependent mode. Although fractionation of lysate does not affect its proteolytic activity toward substrates with free NH2-termini, it completely abolishes the activity toward the blocked substrates, indicating that an important component of the system was either removed or inactivated during fractionation. An NH2-terminal "unblocking" activity that removes the blocking group, thus exposing a free NH2-terminus for recognition according to the N-end rule, does not seem to participate in this pathway. Incubation of whole lysate with labeled histone H2A results in the formation of multiple ubiquitin conjugates. In contrast, the fractionated system is devoid of any significant conjugating activity. These results suggest that a novel conjugating enzyme (possibly a ubiquitin-protein ligase) may be responsible for the degradation of these acetylated proteins by recognizing structural features of the substrate that are downstream and distinct from the NH2-terminal residue.
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