Alfred Lewis Goldberg

Heat shock (HS) promotes protein unfolding, and cells respond by stimulating HS gene expression, ubiquitination of cell proteins, and proteolysis by the proteasome. Exposing HeLa and other cells to 43 °C for 2 h caused a twofold increase in the 26S proteasomes’ peptidase activity assayed at 37 °C. This increase in activity occurred without any change in proteasome amount and did not require new protein synthesis. After affinity-purification from HS cells, 26S proteasomes still hydrolyzed peptides, adenosine 5′-triphosphate, and ubiquitinated substrates more rapidly without any evident change in subunit composition, postsynthetic modification, or association with reported proteasome-activating proteins. After returning HS cells to 37 °C, ubiquitin conjugates and proteolysis fell rapidly, but proteasome activity remained high for at least 16 h. Exposure to arsenite, which also causes proteotoxic stress in the cytosol, but not tunicamycin, which causes endoplasmic reticulum stress, also increased ubiquitin conjugate levels and 26S proteasome activity. Although the molecular basis for the enhanced proteasomal activity remains elusive, we studied possible signaling mechanisms. Proteasome activation upon proteotoxic stress required the accumulation of ubiquitinated proteins since blocking ubiquitination by E1 inhibition during HS or arsenite exposure prevented the stimulation of 26S activity. Furthermore, increasing cellular content of ubiquitin conjugates at 37 °C by inhibiting deubiquitinating enzymes with RA190 or b-AP15 also caused proteasome activation. Thus, cells respond to proteotoxic stresses, apparently in response to the accumulation of ubiquitinated proteins, by activating 26S proteasomes, which should help promote the clearance of damaged cell proteins.

Agents that raise cyclic guanosine monophosphate (cGMP) by activating protein kinase G increase 26S proteasome activities, protein ubiquitination and degradation of misfolded proteins. Therefore, they may be useful in treating neurodegenerative and other diseases caused by an accumulation of misfolded proteins. Mutations in myelin protein zero (MPZ) cause the peripheral neuropathy Charcot-Marie-Tooth type 1B (CMT1B). In peripheral nerves of a mouse model of CMT1B, where the mutant MPZ^S63del is expressed, proteasome activities are reduced, mutant MPZ^S63del and polyubiquitinated proteins accumulate and the unfolded protein response (p-eif2α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZ^S63del, but not of wild-type MPZ. Treating S63del mice with the phosphodiesterase 5 inhibitor, sildenafil—to raise cGMP—increased proteasome activity in sciatic nerves and reduced the levels of polyubiquitinated proteins, the proteasome reporter ubG76V-GFP and p-elF2α. Furthermore, sildenafil treatment reduced the number of amyelinated axons, and increased myelin thickness and nerve conduction velocity in sciatic nerves. Thus, agents that raise cGMP, including those widely used in medicine, may be useful therapies for CMT1B and other proteotoxic diseases.

Although ubiquitination is widely assumed to be the only regulated step in the ubiquitin–proteasome pathway, recent studies have demonstrated several important mechanisms that regulate the activities of the 26S proteasome. Most proteasomes in cells are inactive but, upon binding a ubiquitinated substrate, become activated by a two-step mechanism requiring an association of the ubiquitin chain with Usp14 and then a loosely folded protein domain with the ATPases. The initial activation step is signaled by Usp14’s UBL domain, and many UBL-domain-containing proteins (e.g., Rad23, Parkin) also activate the proteasome. ZFAND5 is a distinct type of activator that binds ubiquitin conjugates and the proteasome and stimulates proteolysis during muscle atrophy. The proteasome’s activities are also regulated through subunit phosphorylation. Agents that raise cAMP and activate PKA stimulate within minutes Rpn6 phosphorylation and enhance the selective degradation of short-lived proteins. Likewise, hormones, fasting, and exercise, which raise cAMP, activate proteasomes and proteolysis in target tissues. Agents that raise cGMP and activate PKG also stimulate 26S activities but modify different subunit(s) and stimulate also the degradation of long-lived cell proteins. Both kinases enhance the selective degradation of aggregation-prone proteins that cause neurodegenerative diseases. These new mechanisms regulating proteolysis thus have clear physiological importance and therapeutic potential.

Significance: Proteasome inhibitors are a highly effective treatment for multiple myeloma, a cancer of plasma cells. These studies were undertaken to understand why myeloma cells are exceptionally sensitive to proteasome inhibition. One proposed explanation is that myeloma proteasomes must continually degrade the large amounts of misfolded immunoglobins these cells produce. If so, myeloma cells should also be unusually sensitive to heat shock, which increases substrate load on 26S proteasomes. Unlike other cells studied, myeloma cell lines undergo extensive apoptosis at 43 °C, and, thus, are highly susceptible to proteotoxic stress. Also, shifting to only 39 °C markedly enhanced their sensitivity to proteasome inhibitors. Thus, mild hyperthermia (e.g. 39 °C) may enhance the efficacy of proteasome inhibitors in the treatment of myeloma.

Significance: Most studies of the regulation of proteolysis by the ubiquitin proteasome system have focused on the control of ubiquitination. However, it is now clear that the activity of the 26S proteasome and rates of protein degradation in cells are also tightly regulated through proteasome phosphorylation. Here we demonstrate that agents that raise cGMP and activate cGMP-dependent protein kinase (e.g., widely used phosphodiesterase 5 inhibitors) stimulate proteasome activities and intracellular proteolysis without affecting autophagy. Furthermore, we showed that raising cGMP reduced the levels of the disease-causing mutant tau in a zebrafish model by increasing its degradation, and also decreased the associated morphological abnormalities. Thus, activating the proteasome via cGMP is a promising strategy to prevent the progression of neurodegenerative diseases.

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure–dynamics–function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod–Wyman–Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis .

Significance: Most protein degradation in eukaryotic cells is catalyzed by the 26S proteasome, which digests proteins marked for destruction by a chain of ubiquitin molecules. Typically, 26S proteasomes are present in cells as inactive particles that can become active when a ubiquitylated substrate binds. Proteasomes are usually viewed as containing a characteristic set of subunits, but, in cells, many important proteins associate transiently with proteasomes to deliver substrates or provide additional activities. Here, we show that a major class of proteasome-binding proteins that contain a ubiquitin-like (Ubl) domain can also enhance the 26S proteasome’s multiple enzymatic activities that are critical for protein degradation. Thus Ubl-containing proteins, in addition to their other functions, have this unexpected regulatory role.

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as novel antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of anti-TB therapeutics that target the Clp system. Here we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Cryo-EM structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that upon binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function, and shows extensive allosteric interactions involving both intra- and inter-ring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis . Significance Statement: The MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis in Mycobacterium tuberculosis , the causative agent of TB. Antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health.