Tricorn protease in bacteria: characterization of the enzyme from Streptomyces coelicolor

Tricorn protease is believed to act downstream of the proteasome, or of other ATP-dependent proteases, cleaving the oligopeptides (mostly 6 to 12 residues) released by them into small peptides (2 to 4 residues), before an array of aminopeptidases finally converts them into free amino acids. Hitherto, the occurrence of Tricorn protease seemed to be limited to some archaea, but genes encoding Tricorn homologs have now been found in several bacterial genomes. Among them is Streptomyces coelicolor A3(2), which has, in fact, two Tricorn-like genes, ScC77.16c and ScE87.19. The proteins encoded by them (TRI-ScC77 and TRI-ScE87) are very similar in their PDZ and TSP domains, but rather divergent in their beta-propeller domains. We have expressed one of them, TRI-ScC77, in E. coil and characterized the recombinant protein structurally and functionally. TRI-ScC77 forms a homohexameric complex of approximately 700 kDa, both in E. coil and in S. coelicolor, with enzymatic properties very similar to the complex from the archaeon Thermoplasma acidophilum. The fact that Tricorn-like proteins exist not only in thermoacidophiles, but also in bacteria inhabiting radically different environments, rules out the possibility that Tricorn protease is an adaptive element that helps to meet the challenges of an extreme habitat.     

Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes

Protein degradation is an essential and strictly controlled process with proteasome and functionally related proteases representing its central part. Tricorn protease (TRI) has been shown to act downstream of the proteasome, degrading produced peptides. Recently, a novel large prokaryotic aminopeptidase oligomeric complex, named TET, has been identified. This complex degrades peptides of different length in organisms where TRI is not present. We determined the crystal structure of TET from the thermophilic archaeon Pyrococcus horikoshii at 1.6 A resolution in native form and in complex with the inhibitor amastatin. We demonstrate that, beside the novel tetrahedral oligomerisation pattern, TET possesses a unique mechanism of substrate attraction and orientation. TET sequentially degrades peptides produced by the proteasome to single amino acids. Furthermore, we reconstituted in vitro the minimal protein degradation system from initial unfolding of labelled protein substrates, up to release of free amino acids. We propose that TET and TRI act as functional analogues in different organisms, with TET being more widely distributed. Thus, TET and TRI represent two evolutionarily diverged pathways of peptide degradation in prokaryotes.     

Purification, crystallization, and preliminary X-ray diffraction analysis of the Tricorn protease hexamer from Thermoplasma acidophilum

Tricorn protease from Thermoplasma acidophilum is a hexameric enzyme; in vivo the hexamers assemble further to form large icosahedral capsids of 14.6 MDa. Recombinant Tricorn protease was purified as an enzymatically active hexamer of 0.72 MDa that formed crystals of octahedral morphology under low-ionic-strength conditions. These crystals belong to space group C2 with unit cell dimensions a = 307.5 A, b = 163.2 A, c = 220.9 A, beta = 105.5 degrees and diffract to 2.2-A resolution using high-brilliance synchrotron radiation. Based on analysis of the self-rotation function and the presence of a pseudo-origin peak in the native Patterson map, a packing model was derived for the complex, comprising 1.5 hexamers per asymmetric unit with a solvent content of 43%. Due to the ninefold noncrystallographic symmetry the Tricorn crystals represent an interesting case for phasing X-ray crystallographic data by electron microscopic phase information.     

Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome

Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the "acidic" and "neutral" chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting that either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) human platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.4; (b) neither the chymotrypsin-like activity of the proteasome, nor proteasome antigens are detected in the cathepsin A preparation; (c) purified proteasome does not exhibit Cbz-Phe-Ala-hydrolyzing activity; (d) Z-lle-Glu-(Ot-Bu)Ala-leucinal (PSI), a compound that selectively inhibits the chymotrypsin-like activity of the proteasome at a concentration of 10 microM has no inhibitory effect on the carboxypeptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, is completely inhibited by micromolar concentrations of lactacystin/beta-lactone. It is therefore concluded that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.     

Impact of ageing on proteasome structure and function in human lymphocytes

Key actors of the immune response, lymphocytes exhibit functional deficits with advancing age. For instance, the age-related decline in lymphocyte proliferation may be related to alteration in the degradation of crucial proteins such as cell-cycle regulators. Degradation of these proteins is mediated by the ubiquitin-26S proteasome system. The proteasome is also the major "housekeeping" proteolytic complex responsible for eliminating intracellular damaged proteins. To investigate the occurrence of proteasome structural and functional age-related alterations, 26S proteasome was purified from peripheral blood lymphocytes of 20-63-year-old donors. Changes in peptidase activity were measured and modifications in the proteasome particle structure were analysed using bi-dimensional electrophoresis. We found the age-related decline of 26S proteasome-specific activity to be associated with an increased yield of post-translational modifications of proteasome subunits, while proteasome content and subunit composition were unchanged. In particular, some catalytic and assembly subunits of the 20S proteasome were preferentially modified with age. Western blotting of proteasome subunits resolved by bi-dimensional electrophoresis showed some of these modified subunits to be glycated, conjugated with a lipid peroxidation product and/or ubiquitinated. In conclusion, it is suggested that structural alterations of proteasome subunits may contribute to the observed decline of proteasome activity with age and could play a major role in immune senescence.     

Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics

Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins—which modulate proteasome activity, stability, localization, or substrate uptake—rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.The proteasome is the proteolytic machinery of the ubiquitin-proteasome system (UPS)¹, the main pathway responsible for degradation of intracellular proteins. As the major cellular protease, the proteasome is a key player in eukaryotic protein homeostasis and dysregulation of the UPS has been involved in neurodegenerative diseases and cancers. Because of this, proteasomes have been identified as therapeutic targets, especially for some cancers (1). Therefore, understanding the structure and function relationship controlling proteasome activity is of major interest in biology.Mammalian proteasomes are composed of a central α7β7β7α7 barrel-shaped catalytic core particle (CP), the 20S proteasome, the structure of which has been determined (2). In cells, the 20S proteasome has been found as an isolated complex, and associated with one or two regulatory particles (RPs) of identical or different protein composition (3). Four RPs have been identified: 19S, PA28αβ, PA28γ, and PA200. The 26S proteasome is a particular complex in which the CP is capped by two 19S RPs, forming a 2.5 MDa complex. Because of a high level of heterogeneity and to the dynamics of the complex, the structure of the mammalian 26S proteasome has yet to be fully determined, but major progress has been made, resulting in a suggested spatial arrangement for the yeast 26S proteasome (4, 5). In the 19S complex, some specific subunits have specialized functions: poly-ubiquitinated (polyUb) substrate recognition, ATP-unfolding, and ubiquitin recycling. These allow ubiquitin-dependent protein degradation. In addition to the RPs, other proteasome interacting proteins (PIPs) bind proteasome complexes and affect their efficiency. These include Ecm29, which plays a role in yeast 26S proteasome assembly and stability (6–8).The CP degrades proteins through three main proteolytic activities, defined as trypsin-like (T-like), chymotrypsin-like (ChT-like), and peptidyl-glutamyl peptide hydrolyzing (PGPH). These activities are exerted by the three beta catalytic subunits, β2, β5, and β1, respectively. An alternative form of the 20S proteasome has been characterized, the immuno-proteasome, where the three standard catalytic subunits are replaced by the so called immuno-subunit counterparts (β2i, β5i, β1i), which can modulate its activity. The proportion of 20S immuno-proteasome varies in different cell types and is increased in cells stimulated by interferon γ (IFNγ) (9, 10). In addition, other 20S proteasome subtypes made up of a mixed assortment of standard catalytic and immuno-subunits were recently described (11). These intermediate 20S proteasome complexes exist in high proportions in many human organs, but also in human tumor cells and dendritic cells. By generating specific antigenic peptides, intermediate 20S proteasome complexes can trigger an immune response (11). Although changes in the CP composition modulate the relative contribution of the cleavage specificity of each catalytic site, overall proteasome activity is drastically increased by association between the CP and RPs.Cell imaging technologies or subcellular fractionation combined with protein blotting techniques have located proteasome complexes in several cellular compartments, mainly the cytosol, nucleus, and associated with the cytoplasmic face of the ER (12). Unlike these antibody-based techniques, quantitative proteomic approaches provide a global view of the cellular distribution of proteins in all their physiological forms (spliced, post-translationally modified, etc.) (13) and have revealed intracellular proteasome relocalization following DNA damage (14). Given the broad function of proteasomes, in quality control, antigenic peptide generation, or short-lived protein-tuned regulation, the cell is likely to adapt proteasome plasticity and dynamics to meet specific subcellular needs or to respond to stress or other stimuli. However, the precise intracellular subunit composition and distribution of proteasome complexes remains largely undetermined. This could be explained by the highly dynamic state of proteasome complexes, their heterogeneity and instability, which make them inherently difficult to study. To deal with this, efficient strategies are needed to purify and quantify fully assembled, active proteasome complexes in homogeneous cellular fractions. These strategies will help us to understand how cells adapt proteasome activity to their needs.In vivo formaldehyde cross-linking can be an efficient tool to study protein–protein interactions and cellular networks (15). It has recently been used to stabilize labile proteasome complexes, allowing the study of the proteasome network in yeast (16) and human cells (17) by quantitative proteomic analyses.In this article, we describe an integrated strategy combining in vivo cross-linking, efficient cell fractionation, affinity purification, and robust label-free quantitative proteomics. We have used this strategy to determine the intracellular distribution of fully assembled active proteasome complexes in human leukemic cells for the first time. Following IFNγ stimulation, our strategy also revealed recruitment of specific PIPs (known to participate in the UPS) to microsomal proteasome complexes. This suggests an important role for these complexes in the endoplasmic reticulum associated degradation (ERAD) pathway.     

Proteasome Activity Is Critical for the cAMP-Induced Differentiation of Neuroblastoma Cells

1. The ubiquitin–proteasome pathway is involved in a variety of cellular functions in mammalian cells. The role of proteasome, however, in the course of cell differentiation is not well characterized. We hypothesized that proteasome activity might be essential during neuronal cell differentiation.2. To investigate the role of proteasome during neuronal differentiation, we made use of a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons upon elevation of the intracellular level of adenosine 3,5-cyclic monophosphate (cAMP). To monitor proteasome activity in NBP2 cells, we integrated an expression cassette~for a short-lived green fluorescent protein (d2EGFP) into these cells, which were designated as NBP2-PN25. When NBP2-PN25 cells were treated with a proteasome inhibitor, lactacystin or MG132, a dose-dependent increase in the constitutive levels of d2EGFP expression was detected.3. We also found that proteasome inhibition by lactacystin during the cAMP-induced differentiation of NBP2-PN25 cells triggered cell death. Both lactacystin and cAMP induction reduced the expression of mRNA for the differentiation-associated genes, such as N-mycand cyclin B1. While cAMP-inducing agents decreased the level of N-myc and cyclin B1 proteins, lactacystin increased the level of these proteins.4. Our data suggest that a reduced level of N-myc and cyclin B1 proteins is critical to commence differentiation, and this can be blocked by a proteasome inhibitor, leading to cell death. Concomitant induction of differentiation and proteasome inhibition, may, therefore, be potentially useful for the treatment of human neuroblastomas.     

PAC1 Gene Knockout Reveals an Essential Role of Chaperone-Mediated 20S Proteasome Biogenesis and Latent 20S Proteasomes in Cellular Homeostasis

The 26S proteasome, a central enzyme for ubiquitin-dependent proteolysis, is a highly complex structure comprising 33 distinct subunits. Recent studies have revealed multiple dedicated chaperones involved in proteasome assembly both in yeast and in mammals. However, none of these chaperones is essential for yeast viability. PAC1 is a mammalian proteasome assembly chaperone that plays a role in the initial assembly of the 20S proteasome, the catalytic core of the 26S proteasome, but does not cause a complete loss of the 20S proteasome when knocked down. Thus, both chaperone-dependent and -independent assembly pathways exist in cells, but the contribution of the chaperone-dependent pathway remains unclear. To elucidate its biological significance in mammals, we generated PAC1 conditional knockout mice. PAC1-null mice exhibited early embryonic lethality, demonstrating that PAC1 is essential for mammalian development, especially for explosive cell proliferation. In quiescent adult hepatocytes, PAC1 is responsible for producing the majority of the 20S proteasome. PAC1-deficient hepatocytes contained normal amounts of the 26S proteasome, but they completely lost the free latent 20S proteasome. They also accumulated ubiquitinated proteins and exhibited premature senescence. Our results demonstrate the importance of the PAC1-dependent assembly pathway and of the latent 20S proteasomes for maintaining cellular integrity.The 26S proteasome is a eukaryotic ATP-dependent protease responsible for the degradation of proteins tagged with polyubiquitin chains (21). The ubiquitin-dependent proteolysis by the proteasome plays a pivotal role in various cellular processes by catalyzing the selective degradation of short-lived regulatory proteins as well as damaged proteins. Thus, the proteasome is essential for the viability of all eukaryotic cells.The 26S proteasome is a large protein complex consisting of two portions; one is the catalytic 20S proteasome of approximately 700 kDa (also called the 20S core particle), and the other is the 19S regulatory particle (RP; also called PA700) of approximately 900 kDa, both of which are composed of a set of multiple distinct subunits (70). The 20S proteasome is a cylindrically shaped stack of four heptameric rings, where the outer and inner rings each are composed of seven homologous α subunits (α1 to α7) and seven homologous β subunits (β1 to β7), respectively (5). The proteolytic active sites reside within the central chamber enclosed by the two inner β-rings, while a small channel formed by the outer α-ring, which is primarily closed, restricts the access of native proteins to the catalytic chamber. Thus, the 20S proteasome is a latent enzyme. Appending 19S RP, which consists of 19 different subunits, to the α-ring enables the 20S proteasome to degrade native proteins; 19S RP accepts ubiquitin chains of substrate proteins, removes ubiquitin chains while unfolding the substrates, and feeds the substrates into the interior proteolytic chamber of the 20S proteasome through the α-ring that is opened when the C-terminal tails of the ATPase subunits of 19S RP are inserted into the intersubunit spaces of the α-ring (24, 62, 74). However, it also has been reported that some denatured or unstructured proteins can be degraded directly by the 20S proteasome even in the absence of 19S RP and ubiquitination (37, 39).Much attention has been focused on how such a highly elaborate structure is achieved. Recent studies have identified various proteasome-dedicated chaperones that assist in the assembly of the proteasome in eukaryotic cells (23, 40, 56, 57, 65, 66). In yeast, while most of the proteasome subunits are essential for viability, the deletion of any of these chaperones does not cause lethality. In fact, many, if not all, of the deletions exhibit subtle phenotypes. In mammalian cells, although the knockdown of the assembly chaperones reduced proteasome assembly and thus proteasome activity, leading to slow cell growth, the degree of reduction was much lower than that which occurred following the knockdown of the proteasome subunit itself (33, 35, 40). These results indicate that the assembly chaperones play an auxiliary role in proteasome biogenesis.Proteasome assembly chaperone 1 (PAC1) is one of the assembly chaperones originally identified in mammalian cells (34). PAC1 plays a role in α-ring formation that occurs during the initial assembly of the 20S proteasome; it also prevents the aberrant dimerization of the α-ring. As is the case for most assembly chaperones, the knockdown of PAC1 in mammalian cells decreases proteasome activity but to a lesser extent than that in, for example, β2 knockdown (34, 35). Therefore, both PAC1-dependent and -independent assembly pathways exist in cells, but the importance of the PAC1-dependent pathway remains elusive. To further elucidate the biological significance of PAC1 and PAC1-dependent proteasome biogenesis, we generated conditional mouse mutants carrying an inactivating mutation in Psmg1, the gene coding for PAC1 protein, in the whole body, the nervous system, and in the liver. Our results demonstrate that PAC1 is essential for the development of a mouse, and that it plays important roles in maintaining cellular integrity in quiescent tissue. Our study revealed for the first time the importance of chaperone-mediated proteasome biogenesis in a whole-body mammalian system and may provide valuable knowledge in medical drug development targeting proteasomes.     

Fellutamide B is a potent inhibitor of the Mycobacterium tuberculosis proteasome

Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K_i = 6.8 nM, whereas it inhibits the human proteasome β5 active site following a two-step mechanism with K_i = 11.5 nM and  = 0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes.     

FV-162 is a novel,orally bioavailable,irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing

Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.The ubiquitin–proteasome system is responsible for the regulation and degradation of the majority of the intracellular proteins in eukaryotic cells.¹ The 26S proteasome is a multi-subunit protein complex that mediates the proteolytic degradation and turnover of damaged, misfolded or excess proteins that have been polyubiquitylated in the cytoplasm and nucleus.^{1, 2} The 26S proteasome consists of a 20S core particle, capped by 19S regulatory particles.^{3, 4} The 19S particle participates in the recognition, processing, unfolding, and translocation of ubiquitylated protein substrates into the 20S core.⁵ Substrates are then degraded inside the chamber of the barrel-like 20S core particle, where the active sites of multiple β1, β2, and β5 subunits catalyze caspase-like (C-L), trypsin-like (T-L), or chymotrypsin-like (CT-L) proteolysis, respectively.^{6, 7} Inhibition of the 26S proteasome activity leads to disruption of the cell cycle and induction of apoptosis.⁸Cancer cells have an increased dependency on the integrity of the ubiquitin–proteasome system machinery compared with normal cells in preclinical studies. This finding is predominantly evident in hematological malignancies, identifying the 26S proteasome as a promising anticancer therapeutic target.^{9, 10, 11, 12} In particular, cells derived from multiple myeloma are notably sensitive to proteasome inhibition, at least in part, owing to their characteristically high rates of immunoglobulin protein biosynthesis and increased proteasome activity.^{13, 14, 15} The continuous activity of the proteasome in myeloma cells makes them particularly susceptible to prolonged inhibition.¹⁶Bortezomib, the first proteasome inhibitor approved for clinical use, is a dipeptide boronic acid that reversibly binds to the active site of the β5 and β1 subunit to competitively inhibit proteasome function.^{9, 10, 17} By inhibiting the proteasome, bortezomib acts through multiple cellular pathways that ultimately result in cell cycle arrest and apoptosis.¹⁸ Bortezomib is currently approved for the treatment of newly diagnosed, relapsed or refractory multiple myeloma and mantle cell lymphoma.¹⁸ Carfilzomib was subsequently developed as a second-generation inhibitor that belongs to the epoxyketone class and irreversibly binds to the active site of the β5 subunit of the proteasome. Carfilzomib is structurally and mechanistically distinct from bortezomib and overcomes bortezomib resistance in multiple myeloma cell lines and in primary multiple myeloma cells from patients.^{17, 19} Carfilzomib is currently also approved for relapsed and refractory multiple myeloma. ONX-0912 (also known as oprozomib) is another epoxyketone class oral proteasome inhibitor that is an analog of carfilzomib.^{20, 21} Similar to carfilzomib, ONX-0912 promotes cell death in primary myeloma cells from patients who relapsed after treatment with bortezomib.²⁰ ONX-0912 has advanced into phase I/II trials in hematological and solid malignancies.^{22, 23, 24}Despite their clinical efficacy, treatment with proteasome inhibitors is associated with a number of toxicities, including neuropathy, thrombocytopenia, and cardiotoxicity.^{25, 26, 27} The toxicity of currently available proteasome inhibitors necessitates administering the drugs in intermittent dosing schedules, typically biweekly. Although intermittent dosing permits proteasome activity in normal tissues during dose holidays, it has been shown to be sub-optimal for therapy in malignant cells.¹⁶ Moreover, infrequent administration at relatively high exposures may give rise to undesirable and potentially unnecessary toxicities in normal cells. Potentially, by moderating exposures, an optimized oral proteasome inhibitor with continuous daily dosing could be developed that exploits the high proteasome dependency in malignant cells while sparing normal cells.In the present study, we report the development of FV-162, a novel, metabolically stable and orally bioavailable epoxyketone-based proteasome inhibitor. FV-162 displays potent anticancer activity and maintains a wide differential activity between malignant and normal cells despite a continuous daily dosing schedule in multiple myeloma cell lines, primary patient cells, and animal models. Overall, our results show that FV-162 inhibits the proteasome, displays metabolic stability, and has a favorable toxicity profile.     

Putting it all together: intrinsic and extrinsic mechanisms governing proteasome biogenesis

Background

The 26S proteasome is at the heart of the ubiquitin-proteasome system, which is the key cellular pathway for the regulated degradation of proteins and enforcement of protein quality control. The 26S proteasome is an unusually large and complicated protease comprising a 28-subunit core particle (CP) capped by one or two 19-subunit regulatory particles (RP). Multiple activities within the RP process incoming ubiquitinated substrates for eventual degradation by the barrel-shaped CP. The large size and elaborate architecture of the proteasome have made it an exceptional model for understanding mechanistic themes in macromolecular assembly.

Objective

In the present work, we highlight the most recent mechanistic insights into proteasome assembly, with particular emphasis on intrinsic and extrinsic factors regulating proteasome biogenesis. We also describe new and exciting questions arising about how proteasome assembly is regulated and deregulated in normal and diseased cells.

Methods

A comprehensive literature search using the PubMed search engine was performed, and key findings yielding mechanistic insight into proteasome assembly were included in this review.

Results

Key recent studies have revealed that proteasome biogenesis is dependent upon intrinsic features of the subunits themselves as well as extrinsic factors, many of which function as dedicated chaperones.

Conclusion

Cells rely on a diverse set of mechanistic strategies to ensure the rapid, efficient, and faithful assembly of proteasomes from their cognate subunits. Importantly, physiological as well as pathological changes to proteasome assembly are emerging as exciting paradigms to alter protein degradation in vivo.

     

Ubiquitin family proteins and their relationship to the proteasome: a structural perspective

Many biological processes rely on targeted protein degradation, the dysregulation of which contributes to the pathogenesis of various diseases. Ubiquitin plays a well-established role in this process, in which the covalent attachment of polyubiquitin chains to protein substrates culminates in their degradation via the proteasome. The three-dimensional structural topology of ubiquitin is highly conserved as a domain found in a variety of proteins of diverse biological function. Some of these so-called "ubiquitin family proteins" have recently been shown to bind components of the 26S proteasome via their ubiquitin-like domains, thus implicating proteasome activity in pathways other than protein degradation. In this chapter, we provide a structural perspective of how the ubiquitin family of proteins interacts with the proteasome.     

Conformational and enzymatic changes of 20S proteasome of rat natural killer cells induced by mono- and divalent cations

We have been investigated the relation between activation of "neutral" and "acidic" chymotrypsin-like (ChT-L) activity and conformational changes in the 20S proteasome complex from the rat natural killer (NK) cells induced by SDS, mono- and divalent cations. The conformational changes were monitored by tryptophan fluorescence and light scattering. It was revealed that the changes in the maximum position and contribution of the short-wavelength spectral component correlated with the alteration of ChT-L activity of the proteasome. Statistical analysis was applied to assign the fluorescence components with tryptophan residues based on the classification of calculated structural parameters of the environment of tryptophan fluorophores in protein. It was proposed that the emission of W13 from [Formula: see text] -subunit located near the cluster of highly conserved proteasome residues is mostly sensitive to the activation of the enzyme. We concluded that the expression of maximal ChT-L activity of 20S proteasome is associated with the conformational changes occurs in this cluster that lead to the proteasome open conformation, allowing substrate access into the proteolytic chamber.     

Identification and localization of a cysteinyl residue critical for the trypsin-like catalytic activity of the proteasome.

Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases.     

Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast.

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.     

Lamotrigine Attenuates Proteasome Inhibition-Induced Apoptosis by Suppressing the Activation of the Mitochondrial Pathway and the Caspase-8- and Bid-Dependent Pathways

Proteasome impairment has been shown to be involved in neuronal degeneration. Antiepileptic lamotrigine has been demonstrated to have a neuroprotective effect. However, the effect of lamotrigine on the proteasome inhibition-induced neuronal cell death has not been studied. Therefore, we assessed the effect of lamotrigine on the proteasome inhibition-induced neuronal cell apoptosis in relation to cell death process using differentiated PC12 cells and SH-SY5Y cells. The proteasome inhibitors MG132 and MG115 induced a decrease in the levels of Bid and Bcl-2 proteins, an increase in the levels of Bax and p53, loss of the mitochondrial transmembrane potential, cytochrome c release and activation of caspases (-8, -9 and -3). The addition of lamotrigine reduced the proteasome inhibitor-induced changes in the apoptosis-related protein levels, production of reactive oxygen species, depletion and oxidation of glutathione (GSH), and cell death in both cell lines. Lamotrigine and N-acetylcysteine alone did not affect the levels of 26S proteasome and activity of 20S proteasome. MG132 did not alter the levels of 26S proteasome but decreased activity of 20S proteasome. Lamotrigine and N-acetylcysteine attenuated MG132-induced decrease in the activity of 20S proteasome. The results show that lamotrigine appears to suppress the proteasome inhibitor-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The suppressive effect of lamotrigine appears to be associated with its inhibitory effect on the production of reactive oxygen species, the depletion and oxidation of GSH and the activity reduction of 20S proteasome.     

Genetic Evidence Linking Age-Dependent Attenuation of the 26S Proteasome with the Aging Process

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies'' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.Ubiquitin-conjugated, misfolded protein aggregates are observed in the brain during normal aging and in late-onset human neurodegenerative diseases, such as Alzheimer''s, Parkinson''s, and polyglutamine diseases (e.g., Huntington''s disease or spinocerebellar ataxias) (9). Many of the mutations that cause dominantly inherited neurodegenerative diseases dramatically increase the amount of protein aggregates in vitro and in vivo, supporting the widely accepted hypothesis that proteotoxicity caused by the aggregates underlies the pathogenesis of many neurodegenerative diseases (32). Proteotoxicity can have many effects, including disruption of microtubule-dependent axonal transport (10), perturbation of membrane permeability (23), and impaired function of the ubiquitin-proteasome system (UPS) (1, 17). Aggregation-mediated toxicity has also been suggested in normal aging, because recent reports show that the impairment of autophagy in the central nervous system causes accumulation of ubiquitinated proteins and leads to neurodegenerative diseases (12, 21). These observations suggest that the continuous clearance of misfolded proteins through cellular degradation systems, including the UPS and autophagy, is important for preventing aggregation-mediated proteotoxicity both in age-related neurodegenerative diseases and in normal aging.Clinical symptoms of neurodegenerative diseases generally do not appear or progress until advanced ages, not only in sporadic forms but also in inherited forms of neurodegenerative diseases (26). These observations suggest that aggregation-mediated toxicity appears in a late-onset manner both in normal aging and in neurodegenerative diseases. Furthermore, a link between the aging process and aggregation-mediated proteotoxicity has been suggested by evidence that Huntington''s disease-associated proteotoxicity was ameliorated when the aging process slowed, that is, the life span extension via decreased insulin/insulin growth factor-1-like signaling in Caenorhabditis elegans (13, 31).A possible mechanism for the late onset of aggregation-mediated toxicity is age-related impairment of the UPS, because loss-of-function mutations in genes encoding UPS components can enhance the cytotoxicity of protein aggregation in dominantly inherited neurodegenerative diseases (4, 5, 18). In addition, an age-related decline of proteasome activity has been observed in the tissues of humans and other mammals (8) and in aged flies (36). Considering the role of the proteasome in neuroprotection and the age dependence of most neurodegenerative diseases, the age-related decline of proteasome activity could well be a key factor both in normal aging and in the late onset and/or progression of neurodegenerative diseases. However, the mechanism underlying the age-related decline of proteasome activity remains to be elucidated, and there is no direct genetic evidence showing that the age-related decline of proteasome activity causes age-related aggregation-mediated toxicity in normal aging and in age-related neurodegenerative diseases.Here, we studied the age-related decline of proteasome activity by using Drosophila melanogaster and found age-related attenuation of the 26S proteasome activity and abundance that was associated with impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we identified Rpn11, which encodes one of the lid subunits in the 19S RP, as a suppressor of the age-dependent progression of a polyglutamine-induced neurodegenerative phenotype. The overexpression of Rpn11 prevented the age-related reduction of the 26S proteasome activity, which suppressed the age-dependent accumulation of ubiquitinated proteins and extended the life span. On the other hand, the loss of function of Rpn11 enhanced the age-related reduction of 26S proteasome activity, leading to a shorter life span, a premature age-dependent accumulation of ubiquitinated proteins, and an early onset of a neurodegenerative phenotype. Our results demonstrate for the first time that the age-related reduction of the 26S proteasome activity is a key factor in the induction of certain age-related biological changes and in the increased risk for the onset or progression of neurodegenerative diseases. Our findings imply that improving the amount and/or activity of the 26S proteasome by overexpressing a lid subunit, such as Rpn11, could provide an extension to the mean life span and prevent the age-dependent onset or progression of neurodegeneration.