Ubiquitin-dependent Degradation of Certain Protein Substrates in Vitro Requires the Molecular Chaperone Hsc70

Abstract

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...

Firefly luciferase mutants as sensors of proteome stress

“…Hsp70 members are highly multifunctional proteins that have been shown to play a key role in proteome maintenance, such as in de novo protein folding (co-or post-translational), protein translocation across membranes (Lyman and Schekman, 1997;Matlack et al, 1999;Young et al, 2003), refolding of stress damaged proteins (Ben-Zvi et al, 2004;Schroder et al, 1993;Sharma et al, 2010), in preventing protein aggregation (Auluck et al, 2002;Broadley and Hartl, 2009;Klucken et al, 2004;Sakahira et al, 2002;Warrick et al, 1999), disaggregation (Ben-Zvi and Goloubinoff, 2001;Diamant et al, 2000;Liberek et al, 2008;Shorter, 2011) and degradation of irreparable misfolded proteins (Bercovich et al, 1997;Fisher et al, 1997;Urushitani et al, 2004). These essential and diverse cellular functions of Hsp70 are attributed to its physical interaction with various co-chaperones such as Hsp40, NEFs and with proteins such as HIP, HOP and CHIP.…”

Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance

“…In the same line, Hsc70 is involved in many basic cellular processes. As a molecular chaperone Hsc70 binds numerous proteins thereby mediating both folding and degradation 269 . One possible approach to study CMA in antigen loading would be the generation of miRNA transgenic animals specific for LAMP2a isoform of the LAMP2 protein, whereby the CMA knock down phenotype could be analyzed.…”

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

“…132 The bound proteins are either renatured by Hsc70, or ubiquitinated and delivered to proteasomes for degradation with the aid of the Hsc70-bound ubiquitin ligase, CHIP, Hsp90 and various other co-chaperones). [136][137][138] Some denatured proteins, in particular the mutant proteins involved in a variety of human neurodegenerative diseases, tend to form large, complex aggregates (aggresomes or inclusion bodies) that may include several ubiquitinated proteins as well as intermediate filaments. 5,139,140 Being too large to be disposed of by the proteasomes, such aggregates are often sequestered by macroautophagy and delivered to the lysosomes.…”