ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

Abstract

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.Energy-dependent proteolysis is responsible for more than 90% of the protein turnover inside the cell (1). This process both removes misfolded and aggregated proteins as part of the response of the cell to stress and controls the concentrations of regulatory proteins (2, 3). In prokaryotes and eukaryotic organelles, energy-dependent proteases fall into five classes as follows: ClpAP, ClpXP, Lon, HslUV (also referred to as ClpYQ), and HflB (also referred to as FtsH). In Archaea, analogous functions are performed by the archaebacterial proteasome, consisting of the proteasome-activating nucleotidase (PAN), 3 working with the 20 S proteasome (4); in the cytoplasm and nucleus of eukaryotes, these same functions are performed by the 26 S proteasome (5). These different proteases show little sequence conservation outside the ATP-binding domains, but they share their overall architecture. They all form oligomeric, barrel-shaped complexes composed of one or more rings with the active sites of proteolysis sequestered inside a central degradation chamber (6). Access channels to these sites are narrow, and proteins have to be unfolded to gain entry (6). Regulatory particles belonging to the AAA family of molecular chaperones assemble on either end of the proteolytic chamber and recognize substrates destined for degradation. After recognition, the regulatory particles translocate the substrate through a central channel to the proteolytic chamber and in doing so unravel folded domains within the substrate. Translocation and unfolding are driven by ATP hydrolysis by the regulatory particles, with conformational changes in the protease transmitted to the substrate by conserved residues in the loops lining the channel (7-10).Protein degradation by AAA proteases is tightly regulated. Most proteins are targeted to ClpAP, ClpXP, HslUV, Lon, HflB, and PAN by sequence motifs in their primary structure (11)(12)(13)(14)(15)(16)(17). Sometimes adaptor proteins recognize and bind sequence elements in substrates and deliver them to the protease, and other times the protease recognizes sequence elements directly (18,19). In contrast,...

Ubiquitinated Proteins Activate the Proteasomal ATPases by Binding to Usp14 or Uch37 Homologs

“…1A) (21) or a coupled pyruvate kinase/lactate dehydrogenase enzymatic assay (Fig. 1B) (22), which gave similar results as reported before (22,24).…”

“…ATP hydrolysis by proteasome particles was measured using the malachite green assay that detects the release of free phosphate (21) or the coupled enzymatic assay using pyruvate kinase and lactate dehydrogenase (22), which assays the production of ADP. ATPase activity and hydrolysis of the small peptide Suc-GGL-amc (Bachem) by proteasomes were measured in the presence of 25 mM Hepes/KOH, pH 8, 2.5 mM MgCl 2 , 125 mM potassium acetate, 0.025% Triton X-100, 1 mM ATP, 1 mM DTT, 0.1 mg/ml BSA (Sigma) as reported previously (11).…”

Dependence of Proteasome Processing Rate on Substrate Unfolding

“…Is that also true of eukaryotic proteasomes? Studies from our laboratory (5,6) and by others (7)(8)(9)(10) are consistent with this conclusion, but studies using purified proteasomes and substrates of well-defined structure have been limited and have not determined the kinetic parameters associated with proteasome action.…”

The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity

“…ATP-dependent proteases in general, and the 26S proteasome in particular, possess an impressive capacity for degrading folded proteins (24). Nonetheless, some proteins are refractory to complete degradation.…”