Alzheimer's associated variant ubiquitin causes inhibition of the 26S proteasome and chaperone expression

Intracellular protein inclusions in Alzheimer's disease and progressive supranuclear palsy contain UBB+1, a variant ubiquitin. UBB+1 is able block the 26S proteasome in cell lines. Proteasome inhibition by drug action has previously been shown to induce a heat-shock response and render protection against stress. We investigated UBB+1 by developing a stable, conditional expression model in SH-SY5Y human neuroblastoma cells. Induction of UBB+1 expression caused proteasome inhibition as was confirmed by reduced ability to process misfolded canavanyl proteins, accumulation of GFPu, a proteasome substrate, and reduced cleavage of a fluorogenic substrate. We show that expression of UBB+1 induces expression of heat-shock proteins. This priming of the chaperone system in these cells promotes a subsequent resistance to tert-butyl hydroperoxide-mediated oxidative stress. We conclude that although UBB+1-expressing cells have a compromised ubiquitin-proteasome system, they are protected against oxidative stress conditions.     

Effects of nucleotides on assembly of the 26S proteasome and degradation of ubiquitin conjugates

We have investigated three aspects of nucleotide usage by the 26S proteasome and its regulatory complex (RC). Both particles hydrolyze the four major ribonucleotides, but ATP and CTP have substantially lower K _s for hydrolysis than do GTP and UTP. The K _ for ATP hydrolysis is 15 m for the 26S proteasome and 30 m for the regulatory complex. Formation of the 26S proteasome from the RC and the 20S proteasome requires about 5 m ATP. Although measurable degradation of Ubiquitin(Ub)-lysozyme conjugates occurs in the presence of CTP, GTP, and UTP, the best nucleotide for Ub-conjugate degradation by the 26S proteasome is ATP, with an estimated K _ of 12 m. In summary, our studies show that micromolar concentrations of ATP are sufficient for several 26S proteasome activities.     

Regulation of ubiquitin and 26S proteasome mediated by phenolic compounds during oxidative stress

Little attention has been devoted to studying the roles of natural antioxidants in the ubiquitin-proteasome pathway during oxidative stress. We demonstrated that a time course revealed that the reassociation of the 19S regulators with the 20S proteasomes occurred automatically and rapidly to reconstitute the 26S proteasomes, with up to 80% completion, within 5 min after H₂O₂ treatment. Ubiquitin, methyl gallate and tannic acid are able to prevent H₂O₂ from inhibiting the 26S activity. We further show that the level of the ubiquitin, S5a and 20S core subunits decreased within 30 min and increased after 24 h of H₂O₂ treatment in Hep-2 cells. Phenolic compounds not only inhibited the 26S activity but also decreased the USP47 levels, which reduce the DNA damage repair rate during oxidative stress; in addition, the presence of DNA fragments, procaspase-3 and a decreased poly (ADP-ribose) polymerase also appeared as a result of the above conditions. Ubiquitin could serve as a protective substrate in H₂O₂ and phenolic compound-treated Hep-2 cells. Methyl gallate and tannic acid, as prooxidants, can attenuate the apoptotic response resulting from long-term oxidative stress. Collectively, these data demonstrate an important role for phenolic compounds in regulating the 26S proteasome and ubiquitin during oxidative stress.     

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.     

Potential roles for ubiquitin and the proteasome during ribosome biogenesis

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.     

VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome

The 26S proteasome recognizes a vast number of ubiquitin-dependent degradation signals linked to various substrates. This recognition is mediated mainly by the stoichiometric proteasomal resident ubiquitin receptors S5a and Rpn13, which harbor ubiquitin-binding domains. Regulatory steps in substrate binding, processing, and subsequent downstream proteolytic events by these receptors are poorly understood. Here we demonstrate that mammalian S5a is present in proteasome-bound and free states. S5a is required for efficient proteasomal degradation of polyubiquitinated substrates and the recruitment of ubiquitin-like (Ubl) harboring proteins; however, S5a-mediated ubiquitin and Ubl binding occurs only on the proteasome itself. We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association. Multiubiquitination events within the VWA domain can further regulate S5a association. Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.     

The ubiquitin/26S proteasome system in plant–pathogen interactions: a never-ending hide-and-seek game

The ubiquitin/26S proteasome system (UPS) plays a central role in plant protein degradation. Over the past few years, the importance of this pathway in plant–pathogen interactions has been increasingly highlighted. UPS is involved in almost every step of the defence mechanisms in plants, regardless of the type of pathogen. In addition to its proteolytic activities, UPS, through its 20S RNase activity, may be part of a still unknown antiviral defence pathway. Strikingly, UPS is not only a weapon used by plants to defend themselves, but also a target for some pathogens that have evolved mechanisms to inhibit and/or use this system for their own purposes. This article attempts to summarize the current knowledge on UPS involvement in plant–microbe interactions, a complex scheme that illustrates the never-ending arms race between hosts and microbes.     

Intracellularly inducible, ubiquitin hydrolase-insensitive tandem ubiquitins inhibit the 26S proteasome activity and cell division

We constructed polyubiquitin derivatives that contain a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. They were designated tandem ubiquitin (tUb) with the number of repeats, such as tUb2. When tUbs were expressed under the control of the GAL1 promoter in the wild-type yeast strain, growth was strongly inhibited. Under these conditions, the degradation of N-end rule substrates, a UFD substrate and Gcn4 was inhibited, indicating that the tUb inhibits 26S proteasome activity. Consistent with this, tUb binds to the 26S proteasome. We showed that tUb inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.     

ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation

Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high-affinity binding of ubiquitin chains, but in their absence, ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2- to 4-fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP), to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded.     

The 26S proteasome: subunits and functions

The 26S proteasome is an eukaryotic ATP-dependent, dumbbell-shaped protease complex with a molecular mass of approximately 2000 kDa. It consists of a central 20S proteasome, functioning as a catalytic machine, and two large V-shaped terminal modules, having possible regulatory roles, composed of multiple subunits of 25–110 kDa attached to the central portion in opposite orientations. The primary structures of all the subunits of mammalian and yeast 20S proteasomes have been determined by recombinant DNA techniques, but structural analyses of the regulatory subunits of the 26S proteasome are still in progress. The regulatory subunits are classified into two subgroups, a subgroup of at least 6 ATPases that constitute a unique multi-gene family encoding homologous polypeptides conserved during evolution and a subgroup of approximately 15 non-ATPase subunits, most of which are structurally unrelated to each other.     

The 26S proteasome: ubiquitin-mediated proteolysis in the tunnel

The 26S proteasome is a self-compartmentalizing protease responsible for the degradation of intracellular proteins. This giant intracellular protease is formed by several subunits arranged into two 19S polar caps-where protein recognition and ATP-dependent unfolding occur-flanking a 20S central barrel-shaped structure with an inner proteolytic chamber. Proteins targeted to the 26S proteasome are conjugated with a polyubiquitin chain by an enzymatic cascade before delivery to the 26S proteasome for degradation into oligopeptides. As a self-compartmentalizing protease, the 26S proteasome circumvents proteins not destined for degradation and can be deployed to the cytoplasmic and nuclear compartments. The 26S proteasome is a representative of emerging group of giant proteases, including tricorn protease, multicorn protease, and TPPII (tripeptidyl peptidase II).     

The 26S proteasome: a dynamic structure

The proteasomal system consists of a proteolytic core, the 20S proteasome, which associates in ATP-dependent and independent reactions with endogenous regulators providing specific substrate binding sites, chaperone function and regulation of activity to the protease. The best known regulators of the 20S proteasome are the 11S and the 19S complexes. Three subunits of the 20S proteasome and the two subunits of the 11S regulator are induced by -Interferon. However, there are no indications for an influence of -interferon on the subunit composition of the 19S regulator and only a few data exist about the dynamics of this complex. The analysis of 19S regulator subunits from yeast mutants reveals that the ATPases appear to be stringently organized in the 26S complex, while peripheral non-ATPases, such as S5a, might serve as subunits which shuttle substrates to the enzyme. A novel non-ATPase has been cloned, sequenced and identified in a complex besides the 19S regulator, the function of which is presently unknown. The dynamic structure of the 26S proteasome is also characterized by transient associations with components such as the modulator and isopeptidases. Certain viral proteins can also be associated with components of the proteasomal system and alter enzymatic activities.     

The 26S proteasome drives trinucleotide repeat expansions

Trinucleotide repeat (TNR) expansion is the causative mutation for at least 17 inherited neurological diseases. An important question in the field is which proteins drive the expansion process. This study reports that the multi-functional protein Sem1 is a novel driver of TNR expansions in budding yeast. Mutants of SEM1 suppress up to 90% of expansions. Subsequent analysis showed that Sem1 facilitates expansions via its function in the 26S proteasome, a highly conserved multi-subunit complex with both proteolytic and non-proteolytic functions. The proteolytic function of the 26S proteasome is relevant to expansions, as mutation of additional proteasome components or treatment of yeast with a proteasome inhibitor suppressed CTG•CAG expansions. The 26S proteasome also drives expansions in human cells. In a human astrocytic cell line, siRNA-mediated knockdown of 26S proteasome subunits PSMC5 or PSMB3 reduced expansions. This expansion phenotype, both in yeast and human cells, is dependent on the proteolytic activity of the proteasome rather than a stress response owing to depletion of free ubiquitin. Thus, the 26S proteasome is a novel factor that drives expansions in both yeast and human cells by a mechanism involving protein degradation.     

Subunit-subunit interactions in the human 26S proteasome

Ubiquitin-dependent proteolysis is mediated by the proteasome. To understand the structure and function of the human 26S proteasome, we cloned complete ORFs of 32 human proteasome subunits and conducted a yeast two-hybrid analysis of their interactions with each other. We observed that there are 114 interacting-pairs in the human 26S proteasome. About 10% (11/114) of these interacting-pairs was confirmed by the GST-pull down analysis. Among these observed interacting subunits, 58% (66/114) are novel and the rest 42% (48/114) has been reported previously in human or in other species. We observed new interactions between the 19S regulatory particle and the beta-rings of the 20S catalytic particle and therefore proposed a modified model of the 26S proteasome.     

Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome

We determined composition and relative roles of deubiquitylating proteins associated with the 26S proteasome in mammalian cells. Three deubiquitylating activities were associated with the 26S proteasome: two from constituent subunits, Rpn11/S13 and Uch37, and one from a reversibly associated protein, Usp14. RNA interference (RNAi) of Rpn11/S13 inhibited cell growth, decreased cellular proteasome activity via disrupted 26S proteasome assembly, and inhibited cellular protein degradation. In contrast, RNAi of Uch37 or Usp14 had no detectable effect on cell growth, proteasome structure or proteolytic capacity, but accelerated cellular protein degradation. RNAi of both Uch37 and Usp14 also had no effect on proteasome structure or proteolytic capacity, but inhibited cellular protein degradation. Thus, proper proteasomal processing of ubiquitylated substrates requires Rpn11 plus either Uch37 or Usp14. Although the latter proteins feature redundant deubiquitylation functions, they also appear to exert noncatalyic effects on proteasome activity that are similar to but independent of one another. These results reveal unexpected functional relationships among multiple deubiquitylating proteins and suggest a model for mammalian 26S proteasome function whereby their concerted action governs proteasome function by linking deubiquitylation to substrate hydrolysis.     

The 26S proteasome of the fission yeast Schizosaccharomyces pombe

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterization of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localization of the 26S proteasome in fission yeast, by immunofluorescence in fixed cells and live cells with the use of a GFP-tagged subunit. Surprisingly, we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. Electron microscopic analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localization showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalization: initially, the signal occupied the area between the dividing nuclei, but at the end of mitosis the signal dispersed, returning to the nuclear periphery on ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.