Proteasome Assembly Chaperone Research Articles

Inhibition of 26S proteasome activity by α-synuclein is mediated by the proteasomal chaperone Rpn14/PAAF1.

Parkinson's disease (PD) is characterized by aggregation of α-synuclein (α-syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α-syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α-syn protein levels and the impact of α-syn on other components of the proteostasis network. Budding yeast Saccharomyces cerevisiae was used as eukaryotic reference organism to study the consequences of α-syn expression on protein dynamics. To address this, we investigated the impact of overexpression of α-syn and S129A variant on the abundance and stability of most yeast proteins using a genome-wide yeast library and a tandem fluorescent protein timer (tFT) reporter as a measure for protein stability. This revealed that the stability of in total 377 cellular proteins was altered by α-syn expression, and that the impact on protein stability was significantly enhanced by phosphorylation at Ser129 (pS129). The proteasome assembly chaperone Rpn14 was identified as one of the top candidates for increased protein stability by expression of pS129 α-syn. Elevated levels of Rpn14 enhanced the growth inhibition by α-syn and the accumulation of ubiquitin conjugates in the cell. We found that Rpn14 interacts physically with α-syn and stabilizes pS129 α-syn. The expression of α-syn along with elevated levels of Rpn14 or its human counterpart PAAF1 reduced the proteasome activity in yeast and in human cells, supporting that pS129 α-syn negatively affects the 26S proteasome through Rpn14. This comprehensive study into the alternations of protein homeostasis highlights the critical role of the Rpn14/PAAF1 in α-syn-mediated proteasome dysfunction.

Normal Proteasome Function Is Needed to Prevent Kidney Graft Injury during Cold Storage Followed by Transplantation.

Kidney transplantation is the preferred treatment for end-stage kidney disease (ESKD). However, there is a shortage of transplantable kidneys, and donor organs can be damaged by necessary cold storage (CS). Although CS improves the viability of kidneys from deceased donors, prolonged CS negatively affects transplantation outcomes. Previously, we reported that renal proteasome function decreased after rat kidneys underwent CS followed by transplantation (CS + Tx). Here, we investigated the mechanism underlying proteasome dysfunction and the role of the proteasome in kidney graft outcome using a rat model of CS + Tx. We found that the key proteasome subunits β5, α3, and Rpt6 are modified, and proteasome assembly is impaired. Specifically, we detected the modification and aggregation of Rpt6 after CS + Tx, and Rpt6 modification was reversed when renal extracts were treated with protein phosphatases. CS + Tx kidneys also displayed increased levels of nitrotyrosine, an indicator of peroxynitrite (a reactive oxygen species, ROS), compared to sham. Because the Rpt6 subunit appeared to aggregate, we investigated the effect of CS + Tx-mediated ROS (peroxynitrite) generation on renal proteasome assembly and function. We treated NRK cells with exogenous peroxynitrite and evaluated PAC1 (proteasome assembly chaperone), Rpt6, and β5. Peroxynitrite induced a dose-dependent decrease in PAC1 and β5, but Rpt6 was not affected (protein level or modification). Finally, serum creatinine increased when we inhibited the proteasome in transplanted donor rat kidneys (without CS), recapitulating the effects of CS + Tx. These findings underscore the effects of CS + Tx on renal proteasome subunit dysregulation and also highlight the significance of proteasome activity in maintaining graft function following CS + Tx.

The prFMNH2-binding chaperone LpdD assists UbiD decarboxylase activation

The UbiD enzyme family of prenylated flavin (prFMN)-dependent reversible decarboxylases is near ubiquitously present in microbes. For some UbiD family members, enzyme activation through prFMNH2-binding and subsequent oxidative maturation of the cofactor readily occurs, both in vivo in a heterologous host and through in vitro reconstitution However, isolation of the active holo-enzyme has proven intractable for others, notably the canonical E. coli UbiD. We show E. coli heterologous expression of the small protein LpdD - associated with the UbiD-like gallate decarboxylase LpdC from Lactobacillus plantarum - unexpectedly leads to 3,4-dihydroxybenzoic acid decarboxylation whole cell activity. This activity was shown to be linked to endogenous E. coli ubiD expression levels. The crystal structure of the purified LpdD reveals a dimeric protein with structural similarity to the eukaryotic heterodimeric proteasome assembly chaperone Pba3/4. Solution studies demonstrate LpdD protein specifically binds to reduced prFMN species only. The addition of the LpdD:prFMNH2 complex supports reconstitution and activation of the purified E. coli apo-UbiD in vitro, leading to modest 3,4-dihydroxybenzoic acid decarboxylation. These observations suggest LpdD acts as a prFMNH2-binding chaperone, enabling apo-UbiD activation through enhanced prFMNH2 incorporation and subsequent oxidative maturation. Hence, while a single highly conserved flavin prenyltransferase UbiX is found associated with UbiD enzymes, our observations suggest considerable diversity in UbiD maturation, ranging from robust autocatalytic to chaperone-mediated processes. Unlocking the full (de)carboxylation scope of the UbiD-enzyme family will thus require more than UbiX co-expression.

Proteasome assembly chaperone translation upon stress requires Ede1 phase separation at the plasma membrane

Proteome adaptation is key to cells surviving stresses. Increased translation of proteasome assembly chaperones (PACs) is critical for increasing proteasome assembly and cell degradative capacity. The endocytic protein Ede1 recruits PAC mRNA to cortical actin patches in Saccharomyces cerevisiae for translation upon stress. We show, through genetic and pharmacological studies, that this is mediated by the capacity of Ede1 to phase separate. PAC expression is maintained when we exchange the phase separating domains from Ede1 for those of unrelated proteins. Without these phase separating regions, PAC expression is not induced upon stress, preventing increased proteasome assembly, causing cell death. This work identifies a mechanism underpinning Ede1-mediated increased translation of specific mRNAs at a time when general translation is repressed.

Wiggle and Shake: Managing and Exploiting Conformational Dynamics during Proteasome Biogenesis.

The 26S proteasome is the largest and most complicated protease known, and changes to proteasome assembly or function contribute to numerous human diseases. Assembly of the 26S proteasome from its ~66 individual polypeptide subunits is a highly orchestrated process requiring the concerted actions of both intrinsic elements of proteasome subunits, as well as assistance by extrinsic, dedicated proteasome assembly chaperones. With the advent of near-atomic resolution cryo-electron microscopy, it has become evident that the proteasome is a highly dynamic machine, undergoing numerous conformational changes in response to ligand binding and during the proteolytic cycle. In contrast, an appreciation of the role of conformational dynamics during the biogenesis of the proteasome has only recently begun to emerge. Herein, we review our current knowledge of proteasome assembly, with a particular focus on how conformational dynamics guide particular proteasome biogenesis events. Furthermore, we highlight key emerging questions in this rapidly expanding area.

UBE4A catalyzes NRF1 ubiquitination and facilitates DDI2-mediated NRF1 cleavage

The transcription factor nuclear factor erythroid 2 like 1 (NFE2L1 or NRF1) regulates constitutive and inducible expression of proteasome subunits and assembly chaperones. The precursor of NRF1 is integrated into the endoplasmic reticulum (ER) and can be retrotranslocated from the ER to the cytosol where it is processed by ubiquitin-directed endoprotease DDI2. DDI2 cleaves and activates NRF1 only when NRF1 is highly polyubiquitinated. It remains unclear how retrotranslocated NRF1 is primed with large amount of ubiquitin and/or very long polyubiquitin chain for subsequent processing. Here, we report that E3 ligase UBE4A catalyzes ubiquitination of retrotranslocated NRF1 and promotes its cleavage. Depletion of UBE4A reduces the amount of ubiquitin modified on NRF1, shortens the average length of polyubiquitin chain, decreases NRF1 cleavage efficiency and causes accumulation of non-cleaved, inactivated NRF1. Expression of a UBE4A mutant lacking ligase activity impairs the cleavage, likely due to a dominant negative effect. UBE4A interacts with NRF1 and the recombinant UBE4A can promote ubiquitination of retrotranslocated NRF1 in vitro. In addition, knocking out UBE4A reduces transcription of proteasomal subunits in cells. Our results indicate that UBE4A primes NRF1 for DDI2-mediated activation to facilitate expression of proteasomal genes.

Prognostic and Immune Infiltration Value of Proteasome Assembly Chaperone (PSMG) Family Genes in Lung Adenocarcinoma

The complexity of lung adenocarcinoma (LUAD) including many interacting biological processes makes it difficult to find therapeutic biomarkers for treatment. Previous studies demonstrated that PSMG (proteasome assembly chaperone) family members regulate the degradation of abnormal proteins. However, transcript expressions of this gene family in LUAD still need to be more fully investigated. Therefore, we used a holistic bioinformatics approach to explore PSMG genes involved in LUAD patients by integrating several high-throughput databases and tools including The Cancer Genome Atlas (TCGA), and Kaplan-Meier plotter database. These data demonstrated that PSMG3 and PSMG4 were expressed at significantly higher levels in neoplastic cells than in normal lung tissues. Notably, increased expressions of these proteins were correlated with poor prognoses of lung cancer patients, which probably confirmed their fundamental roles in the staging of LUAD tumors. Meanwhile, it was also indicated that there were positive correlations between PSMG family genes and the immune response, metabolism of ubiquinone, cell cycle regulatory pathways, and heat shock protein 90 (HSP90)/phosphatidylinositol 3-kinase (PI3K)/Wnt signaling. Experimental data also confirmed that the knockdown of PSMG4 in LUAD cell lines decreased cell proliferation and influenced expressions of downstream molecules. Collectively, this study revealed that PSMG family members are novel prognostic biomarkers for LUAD progression, which also provide new therapeutic targets of LUAD patients.

Independent transcriptomic and proteomic networks reveal common differentially expressed chaperone and interactor genes during tomato cv. Micro-Tom fruit ripening

Heat Shock Proteins (HSPs) are a superfamily of chaperones that have been characterized in different organisms. In plants, HSPs contribute to abiotic stress responses and development. The aim of this work was to perform an integrative analysis using bioinformatic and inferential approaches based on protein-protein interactions (PPI) network building, using transcriptomic and proteomic public data from Solanum lycopersicum cultivar (cv.) Micro-Tom during fruit ripening. We were able to identify specific chaperone networks for each maturing stage at both transcriptional and proteomic levels. Also, we noted that the number of induced chaperones was higher at the transcriptomic level than the proteomic one and increased as the fruit matured. Additionally, we identified clusters at both levels during advanced stages of ripening that were over-represented by HSP families (HSP70 and HSP20), chaperones (Proteasome assembly chaperone) and common protein interactors associated with protein folding, protein synthesis and degradation, and response to stress (Glutathione-S-transferase or GST; Endoplasmic reticulum auxin binding protein ER-ABP; tetratricopeptide repeat protein-ripening regulated protein or TRP-RRP). Finally, we found that some of these up-regulated chaperones show the presence of heat shock element (HSE) motifs in their 5′-untranslated regions (5´UTRs). Our combined inferential bioinformatics approach allowed us to integrate RNA, protein expression and co-expression levels of chaperones involved in cv. Micro-Tom fruit ripening. Statement of significance of the studyThe Chaperone network is largely characterized by diverse families including Heat Shock Proteins (HSPs), which participate in the refolding of denatured proteins in physiological processes such as developmental changes and abiotic stress response. In the present work, we performed the integrative analysis of transcriptomic and proteomic publicly available data from Solanum lycopersicum cv. Micro-Tom to describe interactome networks at both primary stages (transcription and translation) of gene expression in tomato fruits. Our strategy based on interactomics allowed us to identify specific networks at both levels for each maturing stage at fruit ripening, discovering new interactions and characterizing chaperones and co-chaperons forming these complex biological protein networks during tomato fruit ripening.

Oxidative and salt stresses alter the 26S proteasome holoenzyme and associated protein profiles in Arabidopsis thaliana

BackgroundThe 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.ResultsHere, 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.ConclusionsOur 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.

Proteomic analysis revealed the roles of YRR1 deletion in enhancing the vanillin resistance of Saccharomyces cerevisiae

BackgroundVanillin is one of the important phenolic inhibitors in Saccharomyces cerevisiae for bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor gene YRR1 enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion of YRR1 to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms of YRR1 deletion in the protective response of yeast to vanillin.ResultsIn wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP+ and NADH/NAD+ were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed by YRR1 deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation. YRR1 deletion increased the expression of HAA1-encoding transcriptional activator, TMA17-encoding proteasome assembly chaperone and MBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression of HAA1 and TMA17 enhanced resistance to vanillin in S. cerevisiae.ConclusionsCells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response of S. cerevisiae to vanillin and how YRR1 deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of how YRR1 deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

Anaplasma phagocytophilum AptA enhances the UPS, autophagy, and anti-apoptosis of host cells by PSMG3

Anaplasma phagocytophilum is an obligate intracellular bacterium and a common tick-borne infectious pathogen that can cause human granulocytic anaplasmosis (HGA). Effector proteins play an important role in the pathogenic mechanism of A. phagocytophilum, but the specifics of the disease mechanism are unclear. We studied the effector protein AptA (A. phagocytophilum toxin A) using yeast two hybrid assays to screen its interacting protein proteasome assembly chaperone 3 (PSMG3, PAC3), and identified new mechanisms for the pathogenicity of A. phagocytophilum in HEK293T cells. After AptA enters the host cell, it interacts with PSMG3 to enhance the activity of the proteasome, causing ubiquitination and autophagy in the host cell and thereby increasing cross-talk between the ubiquitination-proteasome system (UPS) and autophagy. AptA also reduces the apoptotic efficiency of the host cells. These results offer new clues as to the pathogenic mechanism of A. phagocytophilum and support the hypothesis that AptA interacts with host PSMG3.

Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

Role of a 19S Proteasome Subunit- PSMD10Gankyrin in Neurogenesis of Human Neural Progenitor Cells.

PSMD10Gankyrin, a proteasome assembly chaperone, is a widely known oncoprotein which aspects many hall mark properties of cancer. However, except proteasome assembly chaperon function its role in normal cell function remains unknown. To address this issue, we induced PSMD10Gankyrin overexpression in HEK293 cells and the resultant large-scale changes in gene expression profile were analyzed. We constituted networks from microarray data of these differentially expressed genes and carried out extensive topological analyses. The overrecurring yet consistent theme that appeared throughout analysis using varied network metrics is that all genes and interactions identified as important would be involved in neurogenesis and neuronal development. Intrigued we tested the possibility that PSMD10Gankyrin may be strongly associated with cell fate decisions that commit neural stem cells to differentiate into neurons. Overexpression of PSMD10Gankyrin in human neural progenitor cells facilitated neuronal differentiation via β-catenin Ngn1 pathway. Here for the first time we provide preliminary and yet compelling experimental evidence for the involvement of a potential oncoprotein – PSMD10Gankyrin, in neuronal differentiation.

Dek40 Encodes a PBAC4 Protein Required for 20S Proteasome Biogenesis and Seed Development.

The 26S proteasome, an essential protease complex of the ubiquitin-26S proteasome system (UPS), controls many cellular events by degrading short-lived regulatory proteins marked with polyubiquitin chains. The 20S proteolytic core protease (CP), the catalytic core of the 26S proteasome, is a central enzyme in the UPS. Its biogenesis proceeds in a multistep and orderly fashion assisted by a series of proteasome assembly chaperones. In this study, we identified a novel maize (Zea mays) kernel mutant named defective kernel40 (dek40), which produces small, collapsed kernels and exhibits delayed embryo and endosperm development. Dek40 was identified by map-based cloning and confirmed by transgenic functional complementation. Dek40 encodes a putative cytosol-localized proteasome biogenesis-associated chaperone4 (PBAC4) protein. DEK40 participates in the biogenesis of the 20S CP by interacting with PBAC3. Loss-of-function of DEK40 substantially affected 20S CP biogenesis, resulting in decreased activity of the 26S proteasome. Ubiquitylome analysis indicated that DEK40 influences the degradation of ubiquitinated proteins and plays an essential role in the maintenance of cellular protein homoeostasis. These results demonstrate that Dek40 encodes a PBAC4 chaperone that affects 20S CP biogenesis and is required for 26S proteasome function and seed development in maize.

Molecular and Structural Basis of the Proteasome α Subunit Assembly Mechanism Mediated by the Proteasome-Assembling Chaperone PAC3-PAC4 Heterodimer.

The 26S proteasome is critical for the selective degradation of proteins in eukaryotic cells. This enzyme complex is composed of approximately 70 subunits, including the structurally homologous proteins α1–α7, which combine to form heptameric rings. The correct arrangement of these α subunits is essential for the function of the proteasome, but their assembly does not occur autonomously. Assembly of the α subunit is assisted by several chaperones, including the PAC3-PAC4 heterodimer. In this study we showed that the PAC3-PAC4 heterodimer functions as a molecular matchmaker, stabilizing the α4-α5-α6 subcomplex during the assembly of the α-ring. We solved a 0.96-Å atomic resolution crystal structure for a PAC3 homodimer which, in conjunction with nuclear magnetic resonance (NMR) data, highlighted the mobility of the loop comprised of residues 51 to 61. Based on these structural and dynamic data, we created a three-dimensional model of the PAC3-4/α4/α5/α6 quintet complex, and used this model to investigate the molecular and structural basis of the mechanism of proteasome α subunit assembly, as mediated by the PAC3-PAC4 heterodimeric chaperone. Our results provide a potential basis for the development of selective inhibitors against proteasome biogenesis.

Activating KRAS, NRAS, and BRAF Mutants Enhance Proteasome Capacity and Reduce Endoplasmic Reticulum Stress in Multiple Myeloma, Thereby Promoting Plasma Cell Survival and Proteasome Inhibitor Resistance

Introduction:Multiple myeloma, a malignant proliferation of differentiated plasma cells, is the second most commonly diagnosed hematologic malignancy, and the number of cases may grow by almost 60% between 2010 and 2030. Recent therapeutic advances, including the use of proteasome inhibitors (PIs), have contributed to a doubling of the median overall survival in myeloma patients. This has been paralleled by an increased understanding of the mutational spectrum in this disease, which was first noted almost three decades ago to harbor KRAS and NRAS mutations. KRAS, NRAS, and BRAF mutations which induce p44/42 Mitogen-activated protein kinase (MAPK) signaling are found in about half of myeloma patients, and seem to contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms still remains elusive.Methods:ANBL-6 and U266 human-derived myeloma cell lines have endogenous wild-type (WT) KRAS, NRAS, and BRAF, and were used in this study. All cell lines were validated through The MD Anderson Cancer Center Characterized Cell Line Core Facility. We established lines stably expressing WT, constitutively active (CA)(G12V/G13D/Q61H), or dominant negative (DN)(S17N) KRAS and NRAS mutants, or V600E or DN BRAF. Cell viability was evaluated using the WST-1 tetrazolium reagent, while the chymotrypsin-, trypsin- and caspase-like activities were determined using fluorogenic substrates.Results:CA KRAS, NRAS, and BRAF mutants reduced the sensitivity of ANBL-6 and U266 cells to bortezomib and carfilzomib, while their DN variants sensitized cells to both PIs. This was associated with an induction by these CA mutants of the proteasome chymotrypsin-, trypsin- and caspase-like activities, while the DN variants reduced proteasome activity. These activity changes occurred in parallel with increased expression at both the mRNA and protein levels of catalytically active Proteasome subunit beta (PSMB)-8, PSMB9, and PSMB10, and of the proteasome assembly chaperone Proteasome maturation protein (POMP). Mechanistic studies showed that MAPK induction by the CA mutants caused activation of the ETS transcription factor (ELK1), which was found to have consensus binding sites in the promoters of PSMB8, PSMB9, PSMB10, and POMP. Notably, ELK1 suppression reduced PSMB8, PSMB9, PSMB10, and POMP expression, directly linking RAS/RAF/MAPK signaling to proteasome biology, and this suppression enhanced PI sensitivity. Inhibition of MAPK signaling with either the MAPK kinase (MEK) inhibitor selumetinib or the pan-RAF inhibitor TAK-632 showed synergistic activity with either bortezomib or carfilzomib that was more consistent in cell lines harboring CA mutants as opposed to the DN or WT constructs. Combination regimens of selumetinib or TAK-632 with either bortezomib or carfilzomib induced greater inhibition of the proteasome chymotrypsin-, trypsin- and caspase-like activities than the PIs as single agents. Finally, CA KRAS, NRAS, and BRAF mutants reduced expression levels of genes and proteins involved in the unfolded protein response (UPR), including Activating transcription factor (ATF)-4, -5, and -6, as well as C/EBP homologous protein transcription factor (CHOP) and the spliced variant of X-box binding protein 1 (XBP1s). In contrast, their dominant negative counterparts enhanced expression of the UPR effectors, consistent with an increase in endoplasmic reticulum (ER) stress.Conclusion:Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue that these mutations promote plasma cell survival by reducing cellular stress, thereby distancing myeloma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma. DisclosuresLee:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dick:Takeda Oncology: Employment, Equity Ownership. Chattopadhyay:Takeda Oncology: Employment. Orlowski:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Poseida: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Global profiling of megalocytivirus-induced proteins in tongue sole (Cynoglossus semilaevis) spleen identifies cellular processes essential to viral infection

Megalocytivirus is a DNA virus with a broad host range among farmed fish including tongue sole (Cynoglossus semilaevis). In this study, label-free proteomics was performed to examine protein expression in tongue sole spleen induced by megalocytivirus at 8 and 12 days post infection (dpi). Compared to uninfected control fish, virus-infected fish displayed 315 up-regulated proteins and 111 down-regulated proteins at 8 dpi, and 48 up-regulated proteins and 43 down-regulated proteins at 12 dpi. The expressions of five differentially expressed proteins were confirmed by Western blot. The differentially expressed proteins were enriched in the pathways and processes associated with innate immune response and viral infection. Interference with the expression of two up-regulated proteins of the ubiquitin proteasome system (UPS), i.e. proteasome assembly chaperone 2 and proteasome maturation protein, significantly reduced viral propagation in fish, whereas overexpression of these two proteins significantly enhanced viral propagation. Consistently, inhibition of the functioning of proteasome significantly impaired viral replication in vivo. This study provided the first global protein profile responsive to megalocytivirus in tongue sole, and revealed an essential role of UPS in viral infection.

Synthesis and biological evaluation of thielocin B1 analogues as protein-protein interaction inhibitors of PAC3 homodimer

The synthesis and biological evaluation of thielocin B1 analogues have been demonstrated. Fourteen analogues modified in the central core and terminal carboxylic acid moiety were concisely synthesized by simple esterification or etherification reaction. The evaluation of synthetic analogues as inhibitors of proteasome assembling chaperone (PAC) complexes (the PAC3 homodimer and PAC1/PAC2) revealed that the natural product-like bending structure and terminal carboxylic acid groups were crucial for its biological activity. Moreover, SAR and in silico docking studies indicated that all methyl groups on the diphenyl ether moiety of thielocin B1 contribute to the potent and selective inhibition of the PAC3 homodimer via hydrophobic interactions.

PAC1-PAC2 proteasome assembly chaperone retains the core α4-α7 assembly intermediates in the cytoplasm.

The proteasome core particle (CP) is a cytoplasmic and nuclear protease complex and is comprised of two α-rings and two β-rings stacked in order of αββα. The assembly of CP proceeds by ordered recruitment of β-subunits on an α-ring with help of assembly chaperones PAC1-PAC2, PAC3-PAC4, and UMP1. However, the mechanism of α-ring formation remains unsolved. Here, we show that α4, α5, α6, and α7 form a core intermediate as the initial process of α-ring assembly, which requires PAC3-PAC4. α1 and α3 can be incorporated independently into the core α4-α7 intermediate, whereas α2 incorporation is dependent on preceding incorporation of α1. Through these processes, PAC1-PAC2 prevents nonproductive dimerization of α-ring assembly intermediates. We also found that PAC1-PAC2 overrides the effect of nuclear localization signals of α-subunits and retains α-ring assembly intermediates in the cytoplasm. Our results first show a detailed assembly pathway of proteasomal α-ring and explain the mechanism by which CP assembly occurs in the cytoplasm.

Regulation of proteasome assembly and activity in health and disease.

The proteasome degrades most cellular proteins in a controlled and tightly regulated manner and thereby controls many processes, including cell cycle, transcription, signalling, trafficking and protein quality control. Proteasomal degradation is vital in all cells and organisms, and dysfunction or failure of proteasomal degradation is associated with diverse human diseases, including cancer and neurodegeneration. Target selection is an important and well-established way to control protein degradation. In addition, mounting evidence indicates that cells adjust proteasome-mediated degradation to their needs by regulating proteasome abundance through the coordinated expression of proteasome subunits and assembly chaperones. Central to the regulation of proteasome assembly is TOR complex 1 (TORC1), which is the master regulator of cell growth and stress. This Review discusses how proteasome assembly and the regulation of proteasomal degradation are integrated with cellular physiology, including the interplay between the proteasome and autophagy pathways. Understanding these mechanisms has potential implications for disease therapy, as the misregulation of proteasome function contributes to human diseases such as cancer and neurodegeneration.