The Ubiquitin Proteasome System

Proteins degraded by the ubiquitin/proteasome system are first conjugated to multiple molecules of ubiquitin, a 76-amino acid, 8.5-kDa residue that is highly conserved and present in the cytoplasm of all eukaryotic cells [6]. Ubiquitinated proteins are degraded by the proteolytic 26s proteasome, the catalytic core of which is the 20s proteasome, a barrel-shaped particle consisting of four stacked rings with seven subunits in each ring. This complex and tightly regulated process takes place through different steps (Fig. 1): (1) Ubiquitin is activated in the presence of ATP by ubiquitin-acti-vating enzyme (E1). (2) Activated ubiquitin is transferred from E1 to ubiquitin-conjugating enzyme (E2). (3) The carboxyl group of the activated ubiquitin is coupled to the amino-groups of lysines in the protein substrates by ubiquitin-pro-tein ligase (E3). Reiteration of the ubiquitin-conju-gation reactions creates a chain of five or more ubiquitins linked to each other and then to the protein substrate. (4) The ubiquitinated proteins are unfolded by a 19s complex located on the end of the 20s core proteasome. (5) The unfolded proteins are transported into the central chamber of the 20s core proteasome, where the proteins are cleaved by proteolytic sites located on subunits in the inner rings. The proteins are cut progressively into small peptides of six to twelve amino acids that are subsequently released and rapidly hydrol-ysed to amino acids by cytosolic exopeptidases. (6) The release of ubiquitin from the substrate protein makes ubiquitin available for recycling in the pro-teolytic pathway [6].

Experiments using fluorogenic peptide substrates and inhibitors have defined five activities for the 20s proteasome: (1) a chymotrypsin-like (CTL) activity that cleaves after large hydrophobic

Fig. 1. The ATP-dependent ubiquitin-proteasome pathway. See text for details. UBI, ubiquitin molecule; E1, ubiquitin-acti-vating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase

residues; (2) a trypsin-like (TL) activity that cleaves after basic residues; (3) a post-glutamyl hydrolase (PGP) activity that cleaves after acidic residues; (4) an activity that cleaves preferentially after branched-chain amino acids (BrAAP activity); (5) and an activity that cleaves after small neutral amino acids (SNAAP activity).

E3 ubiquitin-protein ligases are currently believed to be the key component of the conjugation apparatus that confers high specificity to the system. The several hundreds of intracytoplasmic ubiquitin-ligating enzymes, commonly designated as E3s, can be broadly divided into two categories: HECT (homologous to E6-AP C-terminus) domain-containing E3s, and RING (really important new gene)-finger-containing E3s [7]. A critical role in activating proteolysis during atrophy has been ascertained for only three of them, namely, E3[a] and ligases encoded by the genes muscle ring-finger protein-1 (MuRF-1) and muscle atrophy F-box protein (MAFbx), also called atrogin-1 [7] (see below).

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