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Background The 26S proteasome, canonically composed of multi-subunit 19S regulatory (RP) and 20S core (CP) particles, is crucial for cellular proteostasis. Proteasomes are re-modeled, activated, or re-localized and this regulation is critical for plants in response to environmental stresses. The proteasome holoenzyme assembly and dissociation are therefore highly dynamic in vivo. However, the stoichiometric changes of the plant proteasomes and how proteasome associated chaperones vary under common abiotic stresses have not been systematically studied. Results Here, we studied the impact of abiotic stresses on proteasome structure, activity, and interacting partners in Arabidopsis thaliana. We analyzed available RNA expression data and observed that expressions of proteasome coding genes varied substantially under stresses; however, the protein levels of a few key subunits did not change significantly within 24 h. Instead, a switch in the predominant proteasome complex, from 26S to 20S, occurs under oxidative or salt stress. Oxidative stress also reduced the cellular ATP content and the association of HSP70-family proteins to the 20S proteasome, but enhanced the activity of cellular free form CP. Salt stress, on the other hand, did not affect cellular ATP level, but caused subtle changes in proteasome subunit composition and impacted bindings of assembly chaperones. Analyses of an array of T-DNA insertional mutant lines highlighted important roles for several putative assembly chaperones in seedling establishment and stress sensitivity. We also observed that knockout of PBAC1, one of the α-ring assembly chaperones, resulted in reduced germination and tearing of the seed coat following sterilization. Conclusions Our study revealed an evolutionarily conserved mechanism of proteasome regulation during oxidative stress, involving dynamic regulation of the holoenzyme formation and associated regulatory proteins, and we also identified a novel role of the PBAC1 proteasome assembly chaperone in seed coat development.

Targeting the ubiquitin-proteasome system (UPS) - in particular, the proteasome complex - has emerged as an attractive novel cancer therapy. While several proteasome inhibitors have been successfully approved by the Food and Drug Administration for the treatment of hematological malignancies, the clinical efficacy of these inhibitors is unexpectedly lower in the treatment of solid tumors due to the functional and structural heterogeneity of proteasomes in solid tumors. There are ongoing trials to examine the effectiveness of compound and novel proteasome inhibitors that can target solid tumors either alone or in combination with conventional chemotherapeutic agents. The modest therapeutic efficacy of proteasome inhibitors such as bortezomib in solid malignancies demands further research to clarify the exact effects of these proteasome inhibitors on different proteasomes present in cancer cells. The structural, cellular localization and functional analysis of the proteasome complexes in solid tumors originated from different tissues provides new insights into the diversity of proteasomes' responses to inhibitors. In this study, we used an optimized iodixanol gradient ultracentrifugation to purify a native form of proteasome complexes with their intact associated protein partners enriched within distinct cellular compartments. It is therefore possible to isolate proteasome subcomplexes with far greater resolution than sucrose or glycerol fractionations. We have identified differences in the catalytic activities, subcellular distribution, and inhibitor sensitivity of cytoplasmic proteasomes isolated from human colon, breast, and pancreatic cancer cell lines. Our developed techniques and generated results will serve as a valuable guideline for investigators developing a new generation of proteasome inhibitors as an effective targeted therapy for solid tumors.

The 26S proteasome is the major degradation machinery for soluble proteins in eukaryotes. Recent evidence reveals the existence of an alternative ATP-powered protein degradation complex, the Cdc48-20S proteasome complex, and we have identified yeast Sod1, a copper-zinc superoxide dismutase, as an endogenous substrate protein. Here, we identified yeast Ths1, an essential threonyl tRNA synthetase, as another endogenous substrate protein of the Cdc48-20S proteasome. In order to analyze the degradation mechanism in more details, we established an in vitro degradation system reconstituted using purified yeast components. Recombinant Sod1 and Ths1 directly interacted with Cdc48, and were degraded in a Cdc48-20S proteasome-dependent manner. Because the substrate proteins were purified from E. coli cells, no eukaryotic modifications including ubiquitination and phosphorylation exist. Therefore, although the 26S proteasome requires ubiquitination for specific recognition of the substrate proteins, the Cdc48-20S proteasome can degrade a class of substrate proteins without any modifications.

Muscle accretion is affected by the difference between protein synthesis and its degradation. Studies on different species revealed that muscle proteolysis is mediated by different pathways including the ubiquitin-proteasome pathway in which the ubiquitin protein ligases play an important role. These muscle atrophy associated ligases were not well studied in tilapia. In this study, we characterized the ubiquitin protein ligases MuRF1/2/3, Atrogin-1 and F-box25, members of the ubiquitin-proteasome pathway in tilapia, Oreochromis niloticus, and their expressions in the muscle of starved, fed, refed, and control fish. Sequences of these genes revealed presence of Ring finger, B-box, and Cos domains in all MuRF genes, as well as F-box domain in Atrogin-1 and F-box25 genes. Real-time qPCR data analysis showed that expression of MuRF1/2/3, Atrogin-1, F-box25, and proteasome complex genes was significantly upregulated in starved fish compared to fed fish. Concurrently, the proteasome activity was 1.7-folds elevated in the starved fish compared to fed fish. These results confirm the important role of these genes in muscle degradation and suggest potential usage as markers of muscle accretion in tilapia.

Introduction: Multiple myeloma (MM) is the second most commonly diagnosed hematologic malignancy and has an increasing incidence and prevalence globally, and proteasome inhibitors (PIs) form the backbone of some of our most effective regimens for all phases of this disease in fit and frail patients. Areas covered: Strong understanding of the proteasome complex is increasingly important as the rapid development of new PIs and innovative myeloma therapies complicate the use of old and new combination regimens. We focus herein on the pharmacodynamics and pharmacokinetics of the approved PIs and others in development, including their safety and efficacy in corresponding clinical studies. Expert opinion: Advancements such as the first oral PI, ixazomib, with a more convenient route of administration and improved toxicity profile led to an improved quality of life, patient compliance, and all-oral combination regimens which are efficacious for long-term management of standard and high-risk MM. Novel pan-PIs, such as marizomib, hold the promise of superior clinical activity due to irreversible targeting of all multicatalytic proteinase complex subunits. Development of clinically validated biomarkers of PI sensitivity/resistance is required to inform utilization of the most optimal and effective, rationally targeted PI treatments for all MM patients.

Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of age-related neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions.

Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Proteasome inhibitors (PIs) work as the backbone agent and have greatly improved the outcome in majority of newly diagnosed patients with myeloma. However, drug resistance remains the major obstacle causing treatment failure in clinical practice. Indirubin-3’-monoxime (Id-3) is one of the derivatives of Indirubin, a traditional Chinese medicine that has reported to be effective in the treatment of chronic myeloid leukemia and some solid tumors. In this study, we demonstrated the anti-MM activity of Id-3 in both drug sensitive and bortezomib-resistance human MM cell lines and patient-derived cells. Moreover, combination with Id-3 could sensitize MM cells to bortezomib-induced apoptosis. Mechanistically, Id-3 acts as a multifaceted regulator of cell death, which induces DNA damage and cell cycle arrest as well as abrogates NF-κB activation in MM cells. Id-3 efficiently down-regulates USP7 expression, resulting in destabilization of NEK2 and suppression of NF-κB signaling. Importantly, proteasome complex subunits PSME3 (PA28γ) and PSME4 (PA200) were down-regulated in MM cells treated with Id-3. Knockdown of PSME3 or PSME4 by shRNA caused proteasome inhibition and paraprotein accumulation, as well as sensitized MM cells to bortezomib-mediated growth arrest. Clinical data analysis demonstrated that PSME3 and PSME4 are over-expressed in relapsed/refractory MM and associated with inferior overall survival. Taken together, our study indicate that Id-3 is a novel agent triggering proteasome inhibition and represents a promising therapeutic strategy to improve patient outcome in MM. Funding: This work was supported by the Natural Science Foundation of China (8217011925, 81570181, 81920108006), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31006, 2018RC320012), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences CAMS- 2017-I2M-1-005, 2016-I2M-3-023). National Program on Key Basic Research Project 2018ZX09733003. Declaration of Interest: Dr. Kenneth. C. Anderson is the consultant: Pfizer, Amgen, Astrazeneca, Janssen, Precision Biosciences, Mana and Window. Founder/Stock Shareholder: C4 Therapeutics, Oncopep, Raqia and NextRNA. Other authors declared no competing financial interests in relation to the work described. Ethical Approval: The study was approved by the Medical Ethical Committee of the Institute of Hematology & Blood Diseases Hospital, State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China.

Recent advances in next generation sequencing expanded the availability of tumor mutanome data that list the mutations present in cancer cells. Mutated proteins are an interesting source of neoantigens that can be used to specifically target tumor cells in the context of immunotherapy. However, identifying new antigenic peptides from mutated proteins remains challenging. In this chapter, we present Reverse Immunology as an approach to identify potential antigens from any given polypeptide sequence. First, we explain the rationale behind the identification of candidate HLA-binding peptides through mass spectrometry or in silico approaches. Then, we describe the isolation of low-frequency T-cell precursors specific for the candidate peptides using peptide-HLA multimers. Finally, we discuss validation steps leading to the identification of a T-cell clone recognizing tumor cells that endogenously process the candidate peptide. We also present approaches to study the impact of the proteasome complex on candidate peptide processing.

The proteasome complex is mainly responsible for proteolytic degradation of cytosolic proteins, generating the C-terminus of MHC I-restricted peptide ligands and CD8 T cell epitopes. Therefore, prediction of proteasomal cleavage sites is relevant for anticipating CD8 T-cell epitopes. There are two different proteasomes, the constitutive proteasome, expressed in all types of cells, and the immunoproteasome, constitutively expressed in dendritic cells. Although both proteasome forms generate peptides for presentation by MHC I molecules, the immunoproteasome is the main form involved in providing peptide fragments for priming CD8 T cells. On the contrary, the proteasome provides peptides for presentation by MHC I molecules that can be targeted by already primed CD8 T cells. Proteasome cleavage prediction server (PCPS) is a server for predicting cleavage sites generated by both the constitutive proteasome and the immunoproteasome. Here, we illustrate the usage of PCPS to predict proteasome and immunoproteasome cleavage sites and compare the results with those provided by NetChop, a related tool available online. PCPS is implemented for free public use available online at http://imed.med.ucm.es/Tools/pcps/ .

Endometrial cancer incidence increases annually. Several risk factors, including high glucose intake, are associated with endometrial cancer. We investigated whether glucose affects lysine-specific demethylase 1 (LSD1) expression and the responsible molecular mechanisms. A high concentration of glucose stimulated p62 phosphorylation and increased LSD1 protein expression. Knockdown of p62 or treatment with mammalian target of rapamycin (mTOR), transforming growth factor-β activated kinase 1 (TAK1), casein kinase 1 (CK1), and protein kinase C (PKC) inhibitors abrogated glucose-regulated LSD1 expression. Unphosphorylated p62 and LSD1 formed a complex with Kelch-like ECH-associated protein 1 (KEAP1) and were degraded by the KEAP1-dependent proteasome. Phosphorylated p62 increased LSD1 protein expression by escaping the KEAP1 proteasome complex. LSD1 and KEAP1 interaction was enhanced in the presence of the nuclear factor erythroid 2-related factor 2 (NRF2) protein. LSD1 also participated in antioxidant gene regulation with NRF2. In diabetic mice, increasing LSD1and phospho-p62 expression was observed in uterine epithelial cells. Our results indicate that glucose induces p62 phosphorylation through mTOR, TAK1, CK1, and PKC kinases. Subsequently, phospho-p62 competitively interacts with KEAP1 and releases NRF2–LSD1 from the KEAP1 proteasome complex. Our findings may have public health implications for the prevention of endometrial cancer.

The proteasome complex is the main responsible of proteolytic degradation of cytosolic proteins, generating the C-terminus of MHC I-restricted peptide ligands and CD8 T cell epitopes. Prediction of proteasomal cleavage sites is thus relevant for predicting CD8 T cell epitopes. We previously reported an $n$ -gram based method to predict proteasomal cleavage sites named PCPS, which is implemented for free public use online at http://imed.med.ucm.es/pcps/. Here, we used a set of 59 Hepatitis C Virus (HCV) CD8 T-cell epitopes to analyze cleavage predictions by PCPS and compared them with those provided by a related method implemented in NetChop web server. PCPS clearly outperformed NetChop in sensitivity (0.88 and 0.78, respectively); it identified much better than NetChop that the C-terminus of tested CD8 $T$ cell epitopes likely resulted from proteasomal cleavage. However, PCPS identified additional preferential cleavage sites within the tested epitopes more often than NetChop, which resulted in lower specificity (0.57 vs 0.66, respectively). Proteasomal cleavage site predictions are used to enhance CD8 T cell epitopes by discarding peptides resulting from peptide-MHC I binding models that do not have a C-terminus compatible with proteasome production. Thereby, we have now implemented proteasomal cleavage site predictions provided by PCPS $n$ -grams in RANKPEP, a tool to identify peptides presented by MHC molecules, allowing to filter out peptides predicted to bind to MHC I molecules that are unlikely to result from proteasomal cleavage. RANKPEP is available for free public use online at http://imed.med.ucm.es/Tools/rankpep_new.html.

The ubiquitin-proteasome system (UPS) and autophagy are two major degradative pathways of proteins in eukaryotic cells. As about 30% of newly synthesized proteins are known to be misfolded under normal cell conditions, the precise and timely operation of the UPS and autophagy to remove them as well as their tightly controlled regulation, is so important for proper cell function and survival. In the UPS, target proteins are labeled by small proteins called ubiquitin, which are then transported to the proteasome complex for degradation. Alternatively, many greatly damaged proteins are believed to be delivered to the lysosome for autophagic degradation. Although these autophagy and UPS pathways have not been considered to be directly related, many recent studies proposed their close link and dynamic interconversion. In this review, we’ll focus on the several regulatory molecules that function in both UPS and autophagy and their crosstalk. Among the proposed multiple modulators, we will take a closer look at the so-called main connector of UPS-autophagy regulation, p62. Last, the functional role of p62 in the mitophagy and its implication for the pathogenesis of Parkinson''s disease, one of the major neurodegenerative diseases, will be briefly reviewed. [BMB Reports 2020; 53(1): 56-63]

Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. As a single-chain ATP-independent nuclear proteasome activator, proteasome activator 200 (PA200) associates with 20S core particle to form proteasome complex that catalyzes polyubiquitin-independent degradation of acetylated histones, thus playing a pivotal role in DNA repair and spermatogenesis. Here, we present cryo–electron microscopy (cryo-EM) structures of the human PA200-20S complex and PA200 at 2.72 Å and 3.75 Å, respectively. PA200 exhibits a dome-like architecture that caps 20S and uses its C-terminal YYA (Tyr-Tyr-Ala) to induce the α-ring rearrangements and partial opening of the 20S gate. Our structural data also indicate that PA200 has two openings formed by numerous positively charged residues that respectively bind (5,6)-bisdiphosphoinositol tetrakisphosphate (5,6[PP]2-InsP4) and inositol hexakisphosphate (InsP6) and are likely to be the gates that lead unfolded proteins through PA200 and into the 20S. Besides, our structural analysis of PA200 found that the bromodomain (BRD)-like (BRDL) domain of PA200 shows considerable sequence variation in comparison to other human BRDs, as it contains only 82 residues because of a short ZA loop, and cannot be classified into any of the eight typical human BRD families. Taken together, the results obtained from this study provide important insights into human PA200-induced 20S gate opening for substrate degradation and the opportunities to explore the mechanism for its recognition of H4 histone in acetylation-mediated proteasomal degradation., Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. The cryo-EM structures of PA200 and the PA200-20S proteasome complex reveal two openings on PA200 which bind inositol phosphates as cofactors, and novel insights into PA200-induced gate-opening of 20S.

One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K(48)-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

Intraneuronal accumulation of wild‐type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full‐length wild‐type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse‐ and memory‐associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP‐response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ‐20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau‐induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau‐induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ‐20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition., Tau overexpressed decreases the formation of PA28γ‐20S proteasome complex by increasing the association of PKAR2α (an inhibitory subunit of PKA) with PA28γ (a nuclear proteasome activator), which leads to reduced PKAR2α degradation in ubiquitin‐independent proteasome degradation pathway mediated by PA28γ‐20S proteasome complex. PKA inhibition by elevated PKAR2α in nuclear fraction finally induces synapse impairments and memory deficits by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling.

Intracellular signaling mechanisms underlying the opioid system regulation of nociception, neurotransmitters release, stress responses, depression, and the modulation of reward circuitry have been investigated from different points of view. The presence of the ubiquitin proteasome system (UPS) in the synaptic terminations suggest a potential role of ubiquitin-dependent mechanisms in the control of the membrane occupancy by G protein-coupled receptors (GPCRs), including those belonging to the opioid family. In this review, we focused our attention on the role played by the ubiquitination processes and by UPS in the modulation of opioid receptor signaling and in pathological conditions involving the endogenous opioid system. The collective evidence here reported highlights the potential usefulness of proteasome inhibitors in neuropathic pain, addictive behavior, and analgesia since these molecules can reduce pain behavioral signs, heroin self-administration, and the development of morphine analgesic tolerance. Moreover, the complex mechanisms involved in the effects induced by opioid agonists binding to their receptors include the ubiquitination process as a post-translational modification which plays a relevant role in receptor trafficking and degradation. Hence, UPS modulation may offer novel opportunities to control the balance between therapeutic versus adverse effects evoked by opioid receptor activation, thus, representing a promising druggable target.

The β5 subunit of the proteasome has been shown in worms and in human cell lines to be regulatory. In these models, β5 overexpression results in upregulation of the entire proteasome complex which is sufficient to increase proteotoxic stress resistance, improve metabolic parameters, and increase longevity. However, fundamental questions remain unanswered, including the temporal requirements for β5 overexpression and whether β5 overexpression can extend lifespan in other species. To determine if adult-only overexpression of the β5 subunit can increase proteasome activity in a different model, we characterized phenotypes associated with β5 overexpression in Drosophila melanogaster adults. We find that adult-only overexpression of the β5 subunit does not result in transcriptional upregulation of the other subunits of the proteasome as they do in nematodes and human cell culture. Despite this lack of a regulatory role, boosting β5 expression increases the chymotrypsin-like activity associated with the proteasome, reduces both the size and number of ubiquitinated protein aggregates in aged flies, and increases longevity. Surprisingly, these phenotypes were not associated with increased resistance to acute proteotoxic insults or improved metabolic parameters.

The 26S proteasome participates in cell stress responses via its ability to degrade regulatory and damaged proteins. In yeast, mutations in the subunits of the 19S proteasome regulatory subcomplex cause hyper-resistance to 4-nitroquinoline-1-oxide (4-NQO), a chemical mutagen and carcinogen. These data suggest a negative role for the 19S proteasome complex in the cellular response to 4-NQO, although the underlying mechanism is not clear. We proposed that decreased 19S subcomplex activity leads to the stabilisation of Rpn4p, a transcription factor and proteasome substrate. In turn, stabilised Rpn4p may upregulate stress-responsive genes that participate in the response to 4-NQO-induced stress. To test our hypothesis, we impaired the expression of the RPT5 gene, which encodes the ATPase subunit of the 19S subcomplex, by mutating the Rpn4p binding site in its promoter. The mutant strain accumulates polyubiquitinated proteins-a hallmark of compromised proteasome function-and shows hyper-resistance to 4-NQO. We found several groups of genes that conferred resistance to 4-NQO-induced stress and were overexpressed due to the Rpn4p stabilisation and impaired 19S subcomplex function. The upregulated genes are involved in the oxidative and proteotoxic stress response pathways, multidrug resistance and biosynthesis of cysteine and methionine. Consistently, the mutant strain was hyper-resistant to oxidative stress. Our data imply that the ubiquitin-proteasome system may regulate the cellular response to 4-NQO at the transcriptional level.

The Hepatitis C virus (HCV) core protein plays a crucial role in the development of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Its involvement in these diseases is reportedly abolished by a knockout of the proteasome activator PA28γ gene in transgenic mice, suggesting an interaction between the core protein and the PA28γ-proteasome system. This study found a direct interaction between the N-terminal 1–71 fragment of HCV core protein (Core71) and PA28γ in vitro, and that this interaction was found to enhance PA28γ-20S proteasome complex formation. While 20S proteasome activity was increased by PA28γ, it was significantly reduced by Core71 attachment in a dose-dependent manner. These results suggest that the Core-PA28γ interaction has an important role in regulating 20S proteasome activity and furthers our understanding of the pathogenesis of HCV.

Splenomegaly, a major symptom in Plasmodium infection, is extensively studied for its immunopathological role in mice malaria model infected with Plasmodium berghei ANKA. The status of autophagic regulation in hosts in malaria pathogenesis remains unreported till date. This study demonstrated the autophagy, proteasomal degradation and NRF2-KEAP1 antioxidant pathway status in the host during Plasmodium infection taking murine spleen as our organ of interest. Initial staining and autophagic gene expression indicate a possibility of autophagic pathway activation. Although the conversion of LC3A to LC3B and lysosome-autophagosome fusion increases, the final degradation step remains incomplete. Resultant upregulation of p62 and its altered phosphorylated status enhances its binding to keap1 causing NRF2 translocation to the nucleus. NRF2 act as transcription factor upregulating p62 level itself leading to an autoinduction loop of p62 expression. Interestingly, enhancement of P62 interaction with proteasome subunit RPT1 indicates a possible role in transporting ubiquitinated cargo to proteasome complex. Ubiquitination level increased with subsequent upregulation of all three modes of proteasomal degradation i.e trypsin-like, caspase-like and especially chymotrypsin-like. Sqstm1/p62 plays a critical central role in regulating autophagy, proteasomal degradation, and NRF2-KEAP1 pathway. The incomplete autophagic flux in the final step may be a key therapeutic target, as autophagic degradation and subsequent pathogenic peptide presentation is of utmost necessity for downstream immune response.

The 26S proteasome is a multi-subunit protease controlling most of the cytosolic and nuclear protein turnover, regulating many cellular events in eukaryotes. However, functional modification on this complex remains unclear. Here, we showed a novel mechanism that a SUMO ligase AtMMS21 regulates activity of the 26S proteasome in root development of Arabidopsis. Our in vitro and in vivo data supported that AtMMS21 interacts with RPT2a, a subunit of the 26S proteasome. The mutants of AtMMS21 and RPT2a display similar developmental defect of roots, suggesting their association in this process. In addition, RPT2a is modified by SUMO3, potentially related to AtMMS21. During development, the activity of the 26S proteasome is lower in both mutants of AtMMS21 and RPT2a, compared with that of wild type. Furthermore, the protein level but not the RNA level of RPT2a is decreased in the absence of AtMMS21, implying stability regulation of the proteasome complex through the AtMMS21-RPT2a interaction. Taken together, the current study would improve our understanding on the regulatory mechanism of the 26S proteasome via protein modification in root development.

Toxic metal contamination is one of the major environmental concerns of the recent decade, due to the large application of metals in industrial, healthcare and commercial products, even in the form of nanostructures and nanomaterials. Nevertheless, the effects of silver (Ag+) on plants have not yet thoroughly elucidated. Therefore, suspension cell cultures of grapevine were used as a model for investigating silver toxicity. To do this, oxidative stress and programmed cell death (PCD), evaluated as reactive oxygen species production, caspase-3-like activity and ubiquitin-proteasome system, were investigated. As a result, the highest concentration (10 μM) of Ag+ caused a rapid (within 24 h) induction of PCD (approx. 80%), accompanied by generation of reactive oxygen species and activation of caspase-3-like activity. In the presence of specific inhibitor of this enzyme, a partial recovery of cell viability and a strong inhibition of caspase-3-like activity was observed. In addition, silver-induced PCD was accompanied either by increase of poly-ubiquitin conjugated proteins and degradation of subunit PBA1 of the proteasome 20S core, similarly to what found for metal-induced neurotoxicity in animals. The present study shows that silver could induce PCD in grapevine suspension cell cultures, mediated by caspase-3-like activity and oxidative stress. These effects were associated to accumulation of poly-ubiquitin conjugated proteins, suggesting the impairment of ubiquitin-proteasome complex, confirmed by the decrease of the PBA1 subunit. These findings indicate that animal and plant cells could share a common pathway in response to toxic metal, which involves PCD and disassembling of proteasome complex.

Summary Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.

Freeze-thaw stress causes various types of cellular damage, survival and/or proliferation defects, and metabolic alterations. However, the mechanisms underlying how cells cope with freeze-thaw stress are poorly understood. Here, model dough fermentations using two baker's yeast strains, 45 and YF, of Saccharomyces cerevisiae were compared after 2 weeks of cell preservation in a refrigerator or freezer. YF exhibited slow fermentation after exposure to freeze-thaw stress due to low cell viability. A DNA microarray analysis of the YF cells during fermentation revealed that the genes involved in oxidative phosphorylation were relatively strongly expressed, suggesting a decrease in the glycolytic capacity. Furthermore, we found that mRNA levels of the genes that encode the components of the proteasome complex were commonly low, and ubiquitinated proteins were accumulated by freeze-thaw stress in the YF strain. In the cells with a laboratory strain background, treatment with the proteasome inhibitor MG132 or the deletion of each transcriptional activator gene for the proteasome genes ( RPN4 , PDR1 , or PDR3 ) led to marked impairment of model dough fermentation using the frozen cells. Based on these data, proteasomal degradation of freeze-thaw-damaged proteins may guarantee high cell viability and fermentation performance. We also found that the freeze-thaw stress-sensitive YF strain was heterozygous at the PDR3 locus, and one of the alleles (A148T/A229V/H336R/L541P) was shown to possess a dominant negative phenotype of slow fermentation. Removal of such responsible mutations could improve the freeze-thaw stress tolerance and the fermentation performance of baker's yeast strains, as well as other industrial S. cerevisiae strains. IMPORTANCE The development of freezing technology has enabled the long-term preservation and long-distance transport of foods and other agricultural products. Fresh yeast, however, is usually not frozen because the fermentation performance and/or the viability of individual cells is severely affected after thawing. Here, we demonstrate that proteasomal degradation of ubiquitinated proteins is an essential process in the freeze-thaw stress responses of S. cerevisiae . Upstream transcriptional activator genes for the proteasome components are responsible for the fermentation performance after freezing preservation. Thus, this study provides a potential linkage between freeze-thaw stress inputs and the transcriptional regulatory network that might be functionally conserved in higher eukaryotes. Elucidation of the molecular targets of freeze-thaw stress will contribute to advances in cryobiology, such as freezing preservation of human cells, tissues, and embryos for medical purposes and breeding of industrial microorganisms and agricultural crops that adapt well to low temperatures.

The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo.

Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed β-cells evoked by chronic hyperglycemia. Moderate inhibition (10–35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed β-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma β-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in β-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.

Proteins actively participate in a wide range of cellular physiological functions. But aggregation of proteins results in cytotoxicity, and unwanted aggregation of misfolded proteins often causes many diseases. During abnormal protein aggregation events, cells try to cope against such deleterious consequences because of the remarkable functional attempts of two distinct proteolytic mechanisms. These tightly regulative and signaling mechanisms are autophagy pathway and ubiquitin proteasome system. Proteasome complex system holds the elimination capacity of intracellular aberrant protein aggregation. Despite the considerable progress that has been achieved, which elucidates wide function and diverse roles of proteasome system, still several crucial problems remain unanswered. For example, how the complex proteasomes assembly and their interactive pathways determine the precise sense of several proteotoxic insults, which can severely affect the cell survival and homeostasis? The specific degradation of various aberrant proteins that can disturb cellular homeostasis is achieved by proper proteasome functionality, which is yet another unclear and critical challenge. Therefore, a better understanding of the various cellular signaling mechanisms composing the proteasome machinery carries broad therapeutic implications linked with proteopathies. This article signifies the urgent need, which is now crucial for us to improve our understanding of the proteasome architecture, structure, and functions that span multiple level strategies from the molecular level to the cellular level. This systematic in-depth information of proteasome may be helpful in the near future to design a new molecular framework based on intrinsic and extrinsic cellular mechanisms that drive the assembly of proteasome to induce cellular survival against proteostasis imbalance and disease conditions.

Ubiquitin E3 ligases are crucial for eliminating misfolded proteins before they form cytotoxic aggregates that threaten cell fitness and survival. However, it remains unclear how emerging misfolded proteins in the cytoplasm can be selectively recognized and eliminated by E3 ligases in plants. We found that Misfolded Protein Sensing RING E3 ligase 1 (MPSR1) is an indispensable E3 ligase required for plant survival after protein-damaging stress. Under no stress, MPSR1 is prone to rapid degradation by the 26S proteasome, concealing its protein quality control (PQC) E3 ligase activity. Upon proteotoxic stress, MPSR1 directly senses incipient misfolded proteins and tethers ubiquitins for subsequent degradation. Furthermore, MPSR1 sustains the structural integrity of the proteasome complex at the initial stage of proteotoxic stress. Here, we suggest that the MPSR1 pathway is a constitutive mechanism for proteostasis under protein-damaging stress, as a front-line surveillance system in the cytoplasm.

Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

Proteasomes function as the main nonlysosomal machinery of intracellular proteolysis and are involved in the regulation of the majority of important cellular processes. Despite the considerable progress that has been made in understanding the functioning of proteasomes, some issues (in particular, the RNase activity of these ribonucleoprotein complexes and its regulation) remain poorly investigated. In this study, we found to several proteins with electrophoretic mobility that corresponds to that of 20S subunits of the core proteasome complex exhibit endoribonuclease activity with respect to the sense and antisense sequences of the c-myc mRNA 3′-UTR. Mass-spectrometric analysis of tryptic hydrolysates of these proteins showed that the samples contained 20S proteasome subunits—α1 (PSMA6), α5 (PSMA5), α6 (PSMA1), and α7 (PSMA3). A number of new phosphorylation sites of α1 (PSMA6) and α7 (PSMA3) subunits were found, and a form of α5 (PSMA5) subunit with a deletion of 20 N-terminal amino-acid residues was identified. The observed differences in the manifestation of endonuclease activity by individual subunits are apparently due to posttranslational modifications of these proteins (in particular, phosphorylation). It was shown that the specificity of RNase activity changes upon proteasome dephosphorylation and under the influence of Ca2+ and Mg2+ cations. It is concluded that posttranslational modifications of proteasome subunits affect the specificity of their RNase activity.

PSMD9 is a PDZ domain containing chaperone of proteasome assembly. Based on the ability of PDZ-like domains to recognize C-terminal residues in their interactors, we recently predicted and identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as one of the novel interacting partners of PSMD9. Contingent on the reported role of hnRNPA1 in nuclear factor κB (NF-κB) activation, we tested the role of human PSMD9 and hnRNPA1 in NF-κB signaling. We demonstrated in human embryonic kidney 293 cells that PSMD9 influences both basal and tumor necrosis factor α (TNF-α) mediated NF-κB activation through inhibitor of nuclear factor κB α (IκBα) proteasomal degradation. PSMD9 mediates IκBα degradation through a specific domain-motif interaction involving its PDZ domain and a short linear sequence motif in the C-terminus of hnRNPA1. Point mutations in the PDZ domain or deletion of C-terminal residues in hnRNPA1 disrupt interaction between the two proteins which has a direct influence on NF-κB activity. hnRNPA1 interacts with IκBα directly, whereas PSMD9 interacts only through hnRNPA1. Furthermore, hnRNPA1 shows increased association with the proteasome upon TNF-α treatment which has no such effect in the absence of PSMD9. On the other hand endogenous and trans-expressed PSMD9 are found associated with the proteasome complex. This association is unaffected by PDZ mutations or TNF-α treatment. Collectively, these interactions between IκBα, hnRNPA1 and proteasome bound PSMD9 illustrate a potential mechanism by which ubiquitinated IκBα is recruited on the proteasome for degradation. In this process, hnRNPA1 may act as a shuttle receptor and PSMD9 as a subunit acceptor. The interaction sites of PSMD9 and hnRNPA1 may emerge as a vulnerable drug target in cancer cells which require consistent NF-κB activity for survival.

A comparison of different data-independent fragmentation methods combined with liquid chromatography (LC) coupled to high resolution Fourier-transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS) is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complex and their post-translational modifications were identified using a 15-Tesla FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty cycle measurements that better suit on-line LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (Continuous Accumulation of Selected Ions)-CAD. The N-terminus for 9 out of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass measurement accuracy with the LC-FT-ICR system for the 20–30 kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100 kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact protein fragmentation and is an effective addition to the growing inventory of dissociation methods which are compatible with on-line protein separation coupled to FT-ICR MS.

The CAR (constitutive androstane receptor; NR1I3) is a critical xenobiotic sensor that regulates xenobiotic metabolism, drug clearance, energy and lipid homoeostasis, cell proliferation and development. Although constitutively active, in hepatocytes CAR is normally held quiescent through a tethering mechanism in the cytosol, anchored to a protein complex that includes several components, including heat-shock protein 90. Release and subsequent nuclear translocation of CAR is triggered through either direct binding to ligand activators such as CITCO {6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime} or through indirect chemical activation, such as with PB (phenobarbital). In the present study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR's interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR (human CAR). Furthermore, a non-proteolytic function was indicated by its interaction with a SUG1 (suppressor for Gal1), a subunit of the 26S proteasome. Taken together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity.

Biochimica et Biophysica Acta, 1843 (1), ISSN:0006-3002, ISSN:0005-2760, ISSN:0167-4838, ISSN:0304-4157, ISSN:0005-2728, ISSN:0005-2736, ISSN:0167-4781, ISSN:0304-4165, ISSN:0925-4439, ISSN:0167-4889, ISSN:1388-1981, ISSN:1570-9639, ISSN:0304-419X, ISSN:0926-6585, ISSN:0005-2744, ISSN:0926-6593, ISSN:1874-9399, ISSN:0926-6534, ISSN:0005-2787, ISSN:0005-2795, ISSN:0304-4173, ISSN:0926-6577, ISSN:0926-6569, ISSN:0926-6542, ISSN:0926-6526, ISSN:0926

Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson’s disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. We show that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.

In the nervous system, rapidly occurring processes such as neuronal transmission and calcium signaling are affected by short-term inhibition of proteasome function. It is unclear how proteasomes are able to acutely regulate such processes, as this action is inconsistent with their canonical role in proteostasis. Here we describe a mammalian nervous-system-specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is closely associated with neuronal plasma membranes, exposed to the extracellular space, and catalytically active. Selective inhibition of the membrane proteasome complex by a cell-impermeable proteasome inhibitor blocked the production of extracellular peptides and attenuated neuronal-activity-induced calcium signaling. Moreover, we observed that membrane-proteasome-derived peptides were sufficient to induce neuronal calcium signaling. Our discoveries challenge the prevailing notion that proteasomes function primarily to maintain proteostasis, and highlight a form of neuronal communication that takes place through a membrane proteasome complex.

Significance: Alzheimer's disease is a neurodegenerative disorder that is projected to exceed more than 100 million cases worldwide by 2050. Aging is considered the primary risk factor for some 90% of Alzheimer's cases but a significant 10% of patients suffer from aggressive, early-onset forms of the disease. There is currently no effective Alzheimer's treatment and this, coupled with a growing aging population, highlights the necessity to understand the mechanism(s) of disease initiation and propagation. A major hallmark of Alzheimer's disease pathology is the accumulation of amyloid-β (Aβ) aggregates (an early marker of Alzheimer's disease), and neurofibrillary tangles, comprising the hyper-phosphorylated microtubule-associated protein Tau. Recent Advances: Protein oxidation is frequently invoked as a potential factor in the progression of Alzheimer's disease; however, whether it is a cause or a consequence of the pathology is still being debated. The Proteasome complex is a major regulator of intracellular protein quality control and an essential proteolytic enzyme for the processing of both Aβ and Tau. Recent studies have indicated that both protein oxidation and excessive phosphorylation may limit Proteasomal processing of Aβ and Tau in Alzheimer's disease. Critical Issues: Thus, the Proteasome may be a key factor in understanding the development of Alzheimer's disease pathology; however, its significance is still very much under investigation. Future Directions: Discovering how the proteasome is affected, regulated, or dysregulated in Alzheimer's disease could be a valuable tool in the efforts to understand and, ultimately, eradicate the disease. Antioxid. Redox Signal. 25, 886–901.

The development of tumor drug resistance is one of the biggest obstacles on the way to achieve a favorable outcome of chemotherapy. Among various strategies that have been explored to overcome drug resistance, the combination of current chemotherapy with plant polyphenols as a chemosensitizer has emerged as a promising one. Plant polyphenols are a group of phytochemicals characterized by the presence of more than one phenolic group. Mechanistic studies suggest that polyphenols have multiple intracellular targets, one of which is the proteasome complex. The proteasome is a proteolytic enzyme complex responsible for intracellular protein degradation and has been shown to play an important role in tumor growth and the development of drug resistance. Therefore, proteasome inhibition by plant polyphenols could be one of the mechanisms contributing to their chemosensitizing effect. Plant polyphenols that have been identified to possess proteasome-inhibitory activity include (-)-epigallocatechins-3-gallate (EGCG), genistein, luteolin, apigenin, chrysin, quercetin, curcumin and tannic acid. These polyphenols have exhibited an appreciable effect on overcoming resistance to various chemotherapeutic drugs as well as multidrug resistance in a broad spectrum of tumors ranging from carcinoma and sarcoma to hematological malignances. The in vitro and in vivo studies on polyphenols with proteasome-inhibitory activity have built a solid foundation to support the idea that they could serve as a chemosensitizer for the treatment of cancer. In-depth mechanistic studies and identification of optimal regimen are needed in order to eventually translate this laboratory concept into clinical trials to actually benefit current chemotherapy.

J. Neurochem. (2011) 119, 630–643. Abstract Proteasome-mediated proteolysis is a major protein degradation mechanism in cells and its dysfunction has been implicated in the pathogenesis of several neurodegenerative diseases, each with the common features of neuronal death and formation of ubiquitinated inclusions found within neurites, the cell body, or nucleus. Previous models of proteasome dysfunction have employed pharmacological inhibition of the catalytic subunits of the 20S proteasome core, or the genetic manipulation of specific subunits resulting in altered proteasome assembly. In this study, we report the use of dominant negative subunits of the 19S regulatory proteasome complex that mediate the recognition of ubiquitinated substrates as well as the removal of the poly-ubiquitin chain. Interestingly, while each mutant subunit-induced inclusion formation, like that seen with pharmacological inhibition of the 20S proteasome, none was able to induce apoptotic death, or trigger activation of macroautophagy, in either dopaminergic cell lines or primary cortical neurons. This finding highlights the dissociation between the mechanisms of neuronal inclusion formation and the induction of cell death, and represents a novel cellular model for Lewy body-like inclusion formation in neurons.

Proteasome inhibitors have recently emerged as a therapeutic strategy in cancer chemotherapy, but susceptibility to drug resistance limits their efficacy. The marine actinobacterium Salinispora tropica produces salinosporamide A (NPI-0052, marizomib), a potent proteasome inhibitor and promising clinical agent in the treatment of multiple myeloma. Actinobacteria also possess 20S proteasome machinery, raising the question of self-resistance. We identified a redundant proteasome β-subunit, SalI, encoded within the salinosporamide biosynthetic gene cluster and biochemically characterized the SalI proteasome complex. The SalI β-subunit has an altered substrate specificity profile, 30-fold resistance to salinosporamide A, and cross-resistance to the FDA-approved proteasome inhibitor bortezomib. An A49V mutation in SalI correlates to clinical bortezomib resistance from a human proteasome β5-subunit A49T mutation, suggesting that intrinsic resistance to natural proteasome inhibitors may predict clinical outcomes.

Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.

The X protein (HBX) of the hepatitis B virus (HBV) is essential for HBV productive infection in vivo . Our previous study (Z. Hu, Z. Zhang, E. Doo, O. Coux, A. L. Goldberg, and T. J. Liang, J. Virol. 73:7231-7240, 1999) shows that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. Previously, we demonstrated that HBX affects hepadnaviral replication through a proteasome-dependent pathway in cell culture models. In the present study, we studied the effect of the proteasome inhibitor MLN-273 in two HBV mouse models. We demonstrated that administration of MLN-273 to transgenic mice containing the replication-competent HBV genome with the defective HBX gene substantially enhanced HBV replication, while the compound had a minor effect on wild-type HBV transgenic mice. Similar results were obtained by using C57BL/6 mice infected with recombinant adenoviruses expressing the replicating HBV genome. Our data suggest that HBV replication is subjected to regulation by cellular proteasome and HBX functions through the inhibition of proteasome activities to enhance HBV replication in vivo .

KLF1 is an essential erythroid transcription factor (EKLF) involved both in the ß-globin switch from fetal to adult and in definitive erythropoiesis. KLF1 also activates genes encoding heme biosynthetic enzymes, as well as genes involved in the cell cycle and the synthesis of membrane proteins. Heterozygosity for one or several nucleotide changes in KLF1 may be responsible for the so-called dominant Lu(a-b-) phenotype, also known as In(Lu) for "Inhibitor Lutheran" and characterized by a dramatic reduction in the expression levels of the Lutheran blood group antigens and a slight elevation in hemoglobin F (HbF). Several other blood group antigens are also under the direct control of KLF1, but their number and expression level in In(Lu) red blood cells (RBCs) remain a matter of debate. In addition, mutations in KLF1 in humans lead to anemia, acantocytosis and some of these variants, such as CDA type IV, are characterized by ineffective terminal differentiation and defects in enucleation. Given the major role of KLF1 in erythropoiesis, it would not be surprising that In(Lu) RBCs show abnormal properties. In this work, we performed proteomic studies to quantify membrane proteins in In(Lu) vs control RBCs. Results: using label-free mass spectrometry, we analyzed the expression levels of membrane proteins in 5 In(Lu) and 4 control RBCs. Hierarchical clustering allowed to identify two modulation profiles of protein expression (Figure 1). The first profile (red color) is composed of 49 proteins over-expressed in In(Lu) RBCs, with the majority of them corresponding to the "26S proteasome complex" (PSMA, PSMB, PSMD, and PSME). In addition, we confirmed the overexpression of the 26S proteasome complex in In(Lu) RBCs by western blot analyses. The second profile (green color) includes 23 membrane proteins with a lower expression level in In(Lu) RBCs; these proteins correspond to blood group antigens (e.g. Lu/BCAM, CD44), and to cytoskeletal proteins (e.g. dematin, flotillin). Five proteins carrying blood group systems show a decrease of expression in In(Lu) RBCs over 40%: Chido/Rodgers, Xg, Indian, Duffy and Lutheran: antigens. While our results indicating of decreased expression of Indian and Chido/Rodgers systems are consistent with previous reports, the Duffy expression was unexpectedly found to be decreased by 86%. Since we evidenced an important increase in the proteasome complex, and taking into account that KLF1 is involved in the regulation or erythropoiesis, we, investigated the expression profile of the 26 S proteasome expression during erythropoiesis. We observed an important decrease of all proteins of the proteasome complex during erythropoiesis. Conclusion: from our preliminary results reported in this study, we hypothesized that the increase of the 26S proteasome in the In(Lu) individuals may result from defects in organelle loss and could explain the profound dyserytropoiesis observed in the "nan" mice null for KLF1. Figure 1: Comparative proteomics of red blood cell membrane proteins in In(Lu) blood group type. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The 26 S proteasome complex that comprises the 20 S core and 19 S regulatory (with six ATPases) particles is engaged in an ATP-dependent degradation of a variety of key regulatory proteins and, thus, controls important cellular processes. Interestingly, several recent studies have implicated the 19 S regulatory particle in controlling eukaryotic transcriptional initiation or activation independently of the 20 S core particle. However, the mechanism of action of the 19 S proteasome subcomplex in regulation of eukaryotic transcriptional activation is not clearly understood in vivo. Here, using a chromatin immunoprecipitation assay in conjunction with mutational and transcriptional analyses in Saccharomyces cerevisiae, we show that the 19 S proteasomal subcomplex establishes a specific protein interaction network at the upstream activating sequence of the promoter. Such an interaction network is essential for formation of the preinitiation complex at the core promoter to initiate transcription. Furthermore, we demonstrate that the formation of the transcription complex assembly at the promoter is dependent on 19 S ATPase activity. Intriguingly, 19 S ATPases appear to cross-talk for stimulation of the assembly of transcription factors at the promoter. Together, these results provide significant insights as to how the 19 S proteasome subcomplex regulates the formation of the active transcription complex assembly (and, hence, transcriptional initiation) at the promoter in vivo.

Bacteriophage lambda vectors can transfer their genomes into mammalian cells, resulting in expression of phage-encoded genes. However, this process is inefficient. Experiments were therefore conducted to delineate the rate limiting step(s) involved, using a phage vector that contains a mammalian luciferase reporter gene cassette. The efficiency of phage-mediated gene transfer in mammalian cells was quantitated, in the presence or absence of pharmacologic inhibitors of cell uptake and degradation pathways. Inhibitors of lysosomal proteases and proteasome inhibitors strongly enhanced phage-mediated luciferase expression, suggesting that these pathways contribute to the destruction of intracellular phage particles. In contrast, inhibition of endosome acidification had no effect on phage-mediated gene transfer, presumably because phage lambda is tolerant to extended exposure to low pH. These findings provide insights into the pathways by which phage vectors enter and transduce mammalian cells, and suggest that it may be possible to pharmacologically enhance the efficiency of phage-mediated gene transfer in mammalian cells. Finally, the data also suggest that the proteasome complex may serve as an innate defense mechanism that restricts the infection of mammalian cells by diverse viral agents.

The tumour suppressor p33(ING1b) ((ING1b) for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33(ING1b) phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33(ING1b) with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33(ING1b). We found that p33(ING1b) is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33(ING1b). Furthermore, ultraviolet irradiation induces p33(ING1b) phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.

The 26S proteasome plays an essential role in regulating many cellular processes by the degradation of proteins targeted for breakdown by ubiquitin conjugation. The 26S complex is formed from the 20S core, which contains the proteolytic active sites, and 19S regulatory complexes, which bind to the 20S core to activate it and confer specificity for ubiquitinated protein substrates. We have determined the structure of the human 26S proteasome by electron microscopy and single particle analysis. In our reconstructions the crystallographic structure of each of the subunits of the 20S core can be unambiguously docked by direct recognition of each of their densities. Our results show for the first time that binding of the 19S regulatory particle results in the radial displacement of the adjacent subunits of the 20S core leading to opening of a wide channel into the proteolytic chamber. The analysis of a proteasome complex formed from one 20S core with a single 19S regulatory particle attached serve as control to our observations. We suggest locations for some of the 19S regulatory particle subunits.

The life of any protein within a cell begins with transcriptional activation, and ends with proteolytic degradation. Intriguingly, the 26S proteasome complex, a non-lysosomal protein degradation machine comprising the 20S proteolytic core and 19S regulatory particles, has been implicated in intimate regulation of eukaryotic transcriptional activation through diverse mechanisms in a proteolysis-dependent as well as independent manner. Here, we discuss the intricate mechanisms of such proteasomal regulation of eukaryotic gene activation via multiple pathways.

Ubiquitin-proteasome-mediated protein degradation is central to the regulation of many important biological processes, including cell cycle progression, apoptosis and DNA repair. Recognition and degradation of ubiquitinated substrates by the 26S proteasome is tightly regulated to maintain normal cell growth. Disruption of the proteasomal degradation pathway has been implicated in a wide range of human diseases. Although the ubiquitin-proteasome system has been intensively investigated, many key questions remain unanswered in regard to its components and regulation of its activities. A key step towards a full understanding of the pathway is to investigate the proteasome complex subunit composition, heterogeneity, post-translational modifications, assembly, proteasome interaction networks and degradation substrates. Mass spectrometry-based proteomic approaches have been successfully applied for unraveling the details of the proteasome complexes and their substrates in an unprecedented fashion. An overview of the current knowledge of the proteasomal degradation pathway based on mass spectrometry approaches is presented.

ATP binding to the PAN-ATPase complex in Archaea or the homologous 19 S protease-regulatory complex in eukaryotes induces association with the 20 S proteasome and opening of its substrate entry channel, whereas ATP hydrolysis allows unfolding of globular substrates. To clarify the conformational changes associated with ATP binding and hydrolysis, we used protease sensitivity to monitor the conformations of the PAN ATPase from Methanococcus jannischii. Exhaustive trypsin treatment of PAN generated five distinct fragments, two of which differed when a nucleotide (either ATP, ATP gamma S, or ADP) was bound. Surprisingly, the nucleotide concentrations altering protease sensitivity were much lower (K(a) 20-40 microm) than are required for ATP-dependent protein breakdown by the PAN-20S proteasome complex (K(m) approximately 300-500 microm). Unlike trypsin, proteinase K yielded several fragments that differed in the ATP gamma S and ADP-bound forms, and thus revealed conformational transitions associated with ATP hydrolysis. Mapping the fragments generated by each revealed that nucleotide binding and hydrolysis induce local conformational changes, affecting the Walker A and B nucleotide-binding motif, as well as global changes extending to its carboxyl terminus. The location and overlap of the fragments also suggest that the conformation of the six subunits is not identical, probably because they do not all bind ATP simultaneously. Partial nucleotide occupancy was supported by direct assays, which demonstrated that, at saturating conditions, only four nucleotides are bound to hexameric PAN. Using the protease protection maps, we modeled the conformational changes associated with ATP binding and hydrolysis in PAN based on the x-ray structures of the homologous AAA ATPase, HslU.