The Logic of the 26S Proteasome.

The ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation.

Molecular mechanism for activation of the 26S proteasome by ZFAND5.

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated proteins can increase, we studied mouse ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn-finger domain interacts with the Rpt5 ATPase and its C terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate. Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

Immuno- and constitutive proteasomes do not differ in their abilities to degrade ubiquitinated proteins.

Immunoproteasomes are alternative forms of proteasomes that have an enhanced ability to generate antigenic peptides. Recently, Seifert and colleagues reported surprising observations concerning the functions of immunoproteasomes and cellular responses to interferon-γ: (1) that immunoproteasomes degrade ubiquitinated proteins faster than the constitutive proteasomes, (2) that polyubiquitin conjugates accumulate after interferon-γ treatment but then are preferentially degraded by immunoproteasomes, and (3) that immunoproteasome deficiency causes the formation of inclusions and more severe experimental autoimmune encephalomyelitis (EAE). In contrast, we find that polyubiquitin conjugates do not transiently accumulate following IFNγ-treatment and that immunoproteasomes do not prevent the formation of intracellular inclusions or protect against EAE. Furthermore, purified 26S constitutive and immunoproteasomes bind ubiquitin conjugates similarly and degrade them at similar rates. We conclude that, although immunoproteasomes can increase the generation of peptides appropriate for MHC class I presentation, they do not degrade ubiquitinated proteins more efficiently than constitutive particles.

Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.

Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here, we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific α subunit α4 s/PSMA8 and/or the catalytic ß subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis.

Cancer vulnerabilities unveiled by genomic loss.

Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy number losses, we performed integrated analyses of genome-wide copy number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy number loss of that gene. These CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes are enriched for spliceosome, proteasome, and ribosome components. One CYCLOPS gene, PSMC2, encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability.

Maximum entropy determination of mammalian proteome dynamics.

Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

PSMC5 insufficiency and P320R mutation impair proteasome function.

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants.

ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle.

In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules or two ATPγS plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.

A conserved F box regulatory complex controls proteasome activity in Drosophila.

The ubiquitin-proteasome system catalyzes the degradation of intracellular proteins. Although ubiquitination of proteins determines their stabilities, there is growing evidence that proteasome function is also regulated. We report the functional characterization of a conserved proteasomal regulatory complex. We identified DmPI31 as a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a conserved mechanism that is also used by mammalian FBXO7 and PI31. Nutcracker promotes DmPI31 stability, which is necessary for caspase activation, proteasome function, and sperm differentiation. DmPI31 can activate 26S proteasomes in vitro, and increasing DmPI31 levels suppresses defects caused by diminished proteasome activity in vivo. Furthermore, loss of DmPI31 function causes lethality, cell-cycle abnormalities, and defects in protein degradation, demonstrating that DmPI31 is physiologically required for normal proteasome activity.

Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival.

Muscle wasting and cachexia have long been postulated to be key determinants of cancer-related death, but there has been no direct experimental evidence to substantiate this hypothesis. Here, we show that in several cancer cachexia models, pharmacological blockade of ActRIIB pathway not only prevents further muscle wasting but also completely reverses prior loss of skeletal muscle and cancer-induced cardiac atrophy. This treatment dramatically prolongs survival, even of animals in which tumor growth is not inhibited and fat loss and production of proinflammatory cytokines are not reduced. ActRIIB pathway blockade abolished the activation of the ubiquitin-proteasome system and the induction of atrophy-specific ubiquitin ligases in muscles and also markedly stimulated muscle stem cell growth. These findings establish a crucial link between activation of the ActRIIB pathway and the development of cancer cachexia. Thus ActRIIB antagonism is a promising new approach for treating cancer cachexia, whose inhibition per se prolongs survival.

26S proteasomes become stably activated upon heat shock when ubiquitination and protein degradation increase.

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.

Getting to first base in proteasome assembly.

Assembly of complex structures such as the eukaryotic 26S proteasome requires intricate mechanisms that ensure precise subunit arrangements. Recent studies have shed light on the pathway for ordered assembly of the base of the 19S regulatory particle of the 26S proteasome by identifying new precursor complexes and four dedicated chaperones involved in its assembly.

Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation.

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via "shuttling factors." Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum-associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir's ability to enhance cell killing by proteasome inhibitors.

Raising cGMP restores proteasome function and myelination in mice with a proteotoxic neuropathy.

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 MPZS63del is expressed, proteasome activities are reduced, mutant MPZS63del and polyubiquitinated proteins accumulate and the unfolded protein response (p-eif2α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZS63del, 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.

Blocking Cancer Growth with Less POMP or Proteasomes.

Although proteasomes are critical in cell regulation and cancer therapy, little is known about the factors regulating proteasome content or activity. In this issue, Zhang et al. (2015) report that miR-101 suppresses the expression of chaperone POMP and 20S assembly, and certain cancers raise proteasome content by losing miR-101.

Multiple myeloma cells are exceptionally sensitive to heat shock, which overwhelms their proteostasis network and induces apoptosis.

Proteasome inhibitors, such as bortezomib (BTZ), are highly effective and widely used treatments for multiple myeloma. One proposed reason for myeloma cells' exceptional sensitivity to proteasome inhibition is that they produce and continually degrade unusually large amounts of abnormal immunoglobulins. We, therefore, hypothesized that, heat shock may also be especially toxic to myeloma cells by causing protein unfolding, increasing further the substrate load on proteasomes, and, thus, putting further stress on their capacity for protein homeostasis. After a shift from 37 to 43 °C, all four myeloma lines studied underwent extensive apoptosis in 4 h, unlike 13 nonmyeloma cell lines, even though the myeloma cells induced heat-shock proteins and increased protein degradation similar to other cells. Furthermore, two myeloma lines resistant to proteasome inhibitors were also more resistant to 43 °C. Shifting myeloma cells to 43, 41, or 39 °C (which was not cytotoxic) dramatically increased their killing by proteasome inhibitors and inhibitors of ubiquitination or p97/VCP. Combining increased temperature with BTZ increased the accumulation of misfolded proteins and substrate load on the 26S proteasome. The apoptosis seen at 43 °C and at 39 °C with BTZ was mediated by caspase-9 and was linked to an accumulation of the proapoptotic Bcl-2-family member Noxa. Thus, myeloma cells are exceptionally sensitive to increased temperatures, which greatly increase substrate load on the ubiquitin-proteasome system and eventually activate the intrinsic apoptotic pathway. Consequently, for myeloma, mild hyperthermia may be a beneficial approach to enhance the therapeutic efficacy of proteasome inhibitors.

Proteins containing ubiquitin-like (Ubl) domains not only bind to 26S proteasomes but also induce their activation.

During protein degradation by the ubiquitin-proteasome pathway, latent 26S proteasomes in the cytosol must assume an active form. Proteasomes are activated when ubiquitylated substrates bind to them and interact with the proteasome-bound deubiquitylase Usp14/Ubp6. The resulting increase in the proteasome's degradative activity was recently shown to be mediated by Usp14's ubiquitin-like (Ubl) domain, which, by itself, can trigger proteasome activation. Many other proteins with diverse cellular functions also contain Ubl domains and can associate with 26S proteasomes. We therefore tested if various Ubl-containing proteins that have important roles in protein homeostasis or disease also activate 26S proteasomes. All seven Ubl-containing proteins tested-the shuttling factors Rad23A, Rad23B, and Ddi2; the deubiquitylase Usp7, the ubiquitin ligase Parkin, the cochaperone Bag6, and the protein phosphatase UBLCP1-stimulated peptide hydrolysis two- to fivefold. Rather than enhancing already active proteasomes, Rad23B and its Ubl domain activated previously latent 26S particles. Also, Ubl-containing proteins (if present with an unfolded protein) increased proteasomal adenosine 5'-triphosphate (ATP) hydrolysis, the step which commits substrates to degradation. Surprisingly, some of these proteins also could stimulate peptide hydrolysis even when their Ubl domains were deleted. However, their Ubl domains were required for the increased ATPase activity. Thus, upon binding to proteasomes, Ubl-containing proteins not only deliver substrates (e.g., the shuttling factors) or provide additional enzymatic activities (e.g., Parkin) to proteasomes, but also increase their capacity for proteolysis.

cGMP via PKG activates 26S proteasomes and enhances degradation of proteins, including ones that cause neurodegenerative diseases.

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.

An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR.

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 ß-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.

26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation.

Pharmacological agents that raise cAMP and activate protein kinase A (PKA) stimulate 26S proteasome activity, phosphorylation of subunit Rpn6, and intracellular degradation of misfolded proteins. We investigated whether a similar proteasome activation occurs in response to hormones and under various physiological conditions that raise cAMP. Treatment of mouse hepatocytes with glucagon, epinephrine, or forskolin stimulated Rpn6 phosphorylation and the 26S proteasomes' capacity to degrade ubiquitinated proteins and peptides. These agents promoted the selective degradation of short-lived proteins, which are misfolded and regulatory proteins, but not the bulk of cell proteins or lysosomal proteolysis. Proteasome activities and Rpn6 phosphorylation increased similarly in working hearts upon epinephrine treatment, in skeletal muscles of exercising humans, and in electrically stimulated rat muscles. In WT mouse kidney cells, but not in cells lacking PKA, treatment with antidiuretic hormone (vasopressin) stimulated within 5-minutes proteasomal activity, Rpn6 phosphorylation, and the selective degradation of short-lived cell proteins. In livers and muscles of mice fasted for 12-48 hours cAMP levels, Rpn6 phosphorylation, and proteasomal activities increased without any change in proteasomal content. Thus, in vivo cAMP-PKA-mediated proteasome activation is a common cellular response to diverse endocrine stimuli and rapidly enhances the capacity of target tissues to degrade regulatory and misfolded proteins (e.g., proteins damaged upon exercise). The increased destruction of preexistent regulatory proteins may help cells adapt their protein composition to new physiological conditions.