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Peptide:N-glycanase is an evolutionarily conserved deglycosylating enzyme that catalyses the removal of N-linked glycans from cytosolic glycoproteins. Recessive mutations that inactivate this enzyme cause NGLY1 deficiency, a multisystemic disorder with symptoms including developmental delay and defects in cognition and motor control. Developing treatments for NGLY1 deficiency will require an understanding of how failure to deglycosylate NGLY1 substrates perturbs cellular and organismal function. In this review, I highlight insights into peptide:N-glycanase biology gained by studies in the highly tractable genetic model animal Caenorhabditis elegans. I focus on the recent discovery of SKN-1A/Nrf1, an N-glycosylated transcription factor, as a peptide:N-glycanase substrate critical for regulation of the proteasome. I describe the elaborate post-translational mechanism that culminates in activation of SKN-1A/Nrf1 via NGLY1-dependent 'sequence editing' and discuss the implications of these findings for our understanding of NGLY1 deficiency., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome., (© 2022. The Author(s).)

Background: There have been few studies regarding viral involvement in the pathogenesis of renal cell carcinoma (RCC). The aim of this study was to examine the possible association of Epstein-Barr virus (EBV) infection with clinicopathological features and cellular biomarkers including p53, p16INK4a, Ki-67 and nuclear factor-kappa B (NF-κB) in RCC tumors., Methods: In this prospective study, 122 histologically confirmed Formalin-fixed Paraffin-embedded RCC tissue specimens along with 96 specimens of their corresponding peritumoral tissues and 23 samples of blunt renal injuries were subjected to nested polymerase chain reaction (nPCR) in order to amplify EBV DNA sequences. The expression of p53, p16INK4a, Ki-67 and NF-κB was investigated by immunohistochemistry (IHC) assay. Statistical analysis was employed to demonstrate the possible associations., Results: Infection with EBV was found to be significantly associated with RCC. Our results indicate that p65 NF-κB signaling pathway is probably involved in EBV-mediated RCC pathogenesis. Moreover, we found p53, Ki-67 and cytoplasmic NF-κB expression to be associated with tumor nuclear grade in RCC patients. The expression of p53 and Ki-67 was associated with primary tumor category as well. In addition, p53 overexpression was significantly more frequent among nonconventional RCC tumors than the conventional histologic type., Conclusions: Infection with EBV is likely to play an important role in the development of RCC through the constitutive and permanent activation of NF-κB p65 signaling pathway. However, more experiments and supporting data are required to reach a decisive conclusion., (© 2022. The Author(s).)

Background: To identify RA-associated genes and to ascertain epigenetic factors and functional mechanisms underlying RA pathogenesis., Methods: Peripheral blood mononuclear cells (PBMC) transcriptome- and proteome- wide gene expressions were profiled in a case-control study sample. Differentially expressed genes (DEGs) were discovered and validated independently. In-house PBMC genome-wide SNP genotyping data, miRNA expression data and DNA methylation data in the same sample were utilized to identify SNPs [expression quantitative trait locus (eQTLs) and protein quantitative trait locus (pQTLs)], miRNAs, and DNA methylation positions (DMPs) regulating key DEG of interest. Lentivirus transfection was conducted to study the effects of RPN2 on T lymphocyte activation, proliferation, apoptosis, and inflammatory cytokine expression. Rpn2 protein level in plasma was quantitated by ELISA to assess its performance in discriminating RA cases and controls., Results: Twenty-two DEGs were discovered in PBMCs. The most significant DEG, i.e., RPN2, was validated to be up-regulated with RA in PBMCs. A complex regulatory network for RPN2 gene expression in PBMCs was constructed, which consists of 38 eQTL and 53 pQTL SNPs, 3 miRNAs and 2 DMPs. Besides, RPN2 expression was significantly up-regulated with RA in primary T lymphocytes, as well as in PHA-activated T lymphocytes. RPN2 over-expression in T lymphocytes significantly inhibited apoptosis and IL-4 expression and promoted proliferation and activation. PBMCs-expressed RPN2 mRNA and plasma Rpn2 protein demonstrated superior and modest performances in discriminating RA cases and controls, respectively., Conclusions: RPN2 gene influences T lymphocyte growth and activation and is involved in the pathogenesis of RA. Rpn2 may serve as a novel protein biomarker for RA diagnosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes., Competing Interests: Declaration of interests The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratory. K.Mc., R.E.S., and I.M.W. are employees of GeneDx, Inc., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish ( Danio rerio ) from the whole body. The cytosolic fraction of zebrafish hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA), a well-known substrate for the proteasome, in the presence of sodium dodecyl sulfate. From the cytosolic fraction, the 20S proteasome was purified using five column chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S-300 gel, hydroxylapatite, and phenyl Sepharose. Electrophoresis and Western blot analyses showed that zebrafish 20S proteasome subunits have molecular masses ranging from 22 to 33 kDa. The subunit composition of the purified 20S proteasome was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation. Fourteen kinds of 20S subunits were found. As a special characteristic of zebrafish, two proteins of the α1 subunit were identified. In addition, the results suggested that the α8 subunit is in the 20S complex instead of the α4 subunit. In this study, we demonstrated the subunit composition of the 20S proteasome complex present in zebrafish cells.

Objective: Pancreatic cancer (PC) is an extremely malignant tumor with similar morbidity and mortality and lack of an effective treatment. This study explored the prognostic value and molecular mechanisms of proteasome 26S subunit, non-ATPase (PSMD) family genes in pancreatic ductal adenocarcinoma (PDAC). Methods: Survival analyses were performed to elucidate the relationship between prognosis and the level of PSMD expression. ROC curves and nomograms were constructed to predict the prognosis. A bioinformatics analysis was used to explore the co-expression and complex interaction networks of PSMDs. The potential mechanisms were further explored via gene set enrichment analysis (GSEA). Results: We find high levels of PSMD6, PSMD9, PSMD11, and PSMD14 expression were significantly associated with a poorer OS. High PSMD6 and PSMD11 expression was associated with a poorer relapse-free survival (RFS). A risk score model was constructed based on prognosis-related genes. The area under ROC curves (AUC) was 53.3%, 59.3%, and 62.9% for 1-, 2-, 3 years, respectively. Conclusion: GSEA revealed that PSMD6 and PSMD11 play a role in PDAC through various biological processes and signaling pathways, including TP53, CDKN2A, MYC pathway, DNA repair, KRAS, cell cycle checkpoint, NIK, NF-κB signaling pathway, and proteasomes. This study demonstrated that PSMD6 and PSMD11 could serve as a potential prognostic and diagnostic biomarkers for patients with early-stage PDAC after pancreaticoduodenectomy.

Hereditary sensory neuropathy type 1A (HSN1A) is an autosomal, dominantly inherited peripheral neuropathy caused by mutations in serine palmitoyl transferase long chain 1 (SPTLC1), involved in the de novo synthesis of sphingolipids. We have previously reported calcium imbalance, as well as mitochondrial and ER stress in both HSN1 patient lymphoblasts and a transiently transfected cell model. In this study, we investigated the role of the Ca 2+ -activated protease calpain in destabilizing the cell cytoskeleton, by examining calpain activity in SH-SY5Y cells overexpressing the V144D mutant and changes in microtubule-associated proteins (MAP). Intramitochondrial Ca 2+ was found to be significantly depleted and cytoplasmic Ca 2+ increased in the V144D mutant. Subsequently, calpain and proteasome activity were increased and calpain substrates, microtubule associated proteins MAP2, and tau were significantly reduced in the microtubule fraction of the mutant. Significant changes were also found in motor proteins dynein and KIF2A detected in the microtubule fraction of cells overexpressing the V144D mutation. There was also a reduction in anterograde and retrograde mitochondrial transport velocities in the V144D mutant. These findings strongly implicate cytoskeletal aberration caused by Ca 2+ dysregulation and subsequent loss of microtubule transport functions as the cause of axonal dying back that is characteristic of HSN1.

PSMA3, a member of the proteasome subunit, has been shown to play a major player in protein degradation. Reportedly, PSMA3 functions as a negative regulator in various cancers including colon, pancreatic and gastric cancers. However, the contributions of PSMA3 to the progression of esophageal squamous cell carcinoma (ESCC) and the underlying mechanism remain unclear. Therefore, in this study, we investigated whether PSMA3 is involved in ESCC progression and the potential underlying mechanism. The results revealed that PSMA3 was highly expressed in the ESCC tumor tissues and functioned as a negative indicator according to the data from The Cancer Genome Atlas (TCGA)/Gene Expression Omnibus (GEO) datasets and clinical patients' samples. Pathway enrichment analysis showed that PSMA3 was closely correlated with ESCC cancer stemness and the inflammatory response; however, this correlation was absent after knockdown of PSMA3 in vitro. We further demonstrated that PSMA3 suppressed CD8 + T-cells infiltration depending on the C-C motif chemokine ligand 3 (CCL3)/C-C motif chemokine receptor 5 (CCR5) axis. Collectively, these results demonstrate the role of PSMA3 in ESCC cancer stemness and the negative regulation of CD8 T-cells infiltration mediated by PSMA3. The results of this study may provide a potential target for the immuno-oncology effect of PSMA3 in ESCC therapy., (© 2021 Wiley-VCH GmbH.)

Nitric oxide (NO) is a versatile signaling molecule with diverse roles in plant biology. The NO-mediated signaling mechanism includes post-translational modifications (PTMs) of target proteins. There exists a close link between NO-mediated PTMs and the proteasomal degradation of proteins via ubiquitylation. In some cases, ubiquitin-mediated proteasomal degradation of target proteins is followed by an NO-mediated post-translational modification on them, while in other cases NO-mediated PTMs can regulate the ubiquitylation of the components of ubiquitin-mediated proteasomal machinery for promoting their activity. Another pathway that links NO signaling with the ubiquitin-mediated degradation of proteins is the N-degron pathway. Overall, these mechanisms reflect an important mechanism of NO signal perception and transduction that reflect a close association of NO signaling with proteasomal degradation via ubiquitylation. Therefore, this review provides insight into those pathways that link NO-PTMs with ubiquitylation.

The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits (Erzberger and Berger, 2006; Puchades et al., 2020). How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is unknown. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome's heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions are consistent with a wide range of experimental observations in this and previous studies and suggested novel mechanistic features of the proteasomal ATPases. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding status. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex., Competing Interests: RF, JH, MZ, YL No competing interests declared, (© 2022, Fang et al.)

Osteoporosis (OP) is characterized by decreased bone mineral density and impaired bone strength. Carfilzomib (CFZ) is a new-generation proteasome inhibitor and has been found to affect bone metabolism. However, the effect and mechanism of CFZ on OP has not been investigated systematically. In this study, we found that protein levels of proteasome activator subunit 1/2 (PSME1/2) increased in OP, and accumulated mostly in osteoblasts and osteoclasts. Treatment with PSME1/2 recombinant protein inhibited osteogenesis and promoted osteoclast formation in vitro. Also, PSME1/2 inhibited the expression of β-catenin protein, resulting in limitation of Wnt/β-catenin signaling. CFZ inhibited PSME1 and PSME2 proteasome activities and increased β-catenin protein level, resulting in the translocation of β-catenin to the nucleus and activation of canonical Wnt/β-catenin signaling, further promoting osteogenesis and inhibiting osteoclastic differentiation. In vivo, we conducted ovariectomy (OVX) to create a model of OVX-induced postmenopausal OP in mice. When analyzed by micro-CT scanning, enhancement of bone mineral density, bone volume, trabecular number, and thickness was seen in the CFZ-treated mice. Also, we noticed increased osteogenesis and decreased osteoclastogenesis, diminished expression of PSME1 and PSME2 and activated Wnt/β-catenin signaling in bone sections from OP mice treated with CFZ. Overall, our data indicated that PSME1/2 may serve as new targets for the treatment of OP, and targeting PSME1/2 with CFZ provides a candidate therapeutic molecule for postmenopausal OP., (Copyright © 2021 Elsevier B.V. All rights reserved.)

de Almeida et al. (2021) developed a temporally controlled CRISPR-Cas9 screen to identify mechanisms controlling MYC levels and discovered that intact proteasomes are imported into the nucleus by AKIRIN2 binding to proteasomes at one end and a nuclear import receptor at the other., (Published by Elsevier Inc.)

Protein aggregation and degradation via autophagy (aggrephagy) are major strategies adopted by cells to remove misfolded polypeptides when there is proteasome dysfunction. The functional protein complex consisting of heat shock protein 70 (Hsp70), cochaperone ubiquitin ligase carboxyl-terminal of Hsp70/Hsp90 interacting protein (CHIP), and co-chaperone Bcl-2-associated athanogene 3 (BAG3) has been associated with the activation of protein aggregation. However, data on the mechanisms of action of the complex in the protein degradation remains scant. Here, we report that upon proteasome stress, the M2 isoform of pyruvate kinase (PKM2) promotes the aggregation of ubiquitinated proteins and its knockout or knockdown aggravates the sensitivity of cells to proteasome inhibitors. Besides, following proteasome inhibition, PKM2 promotes the interaction of BAG3 with CHIP and HSP70. Interestingly, re-expression of loss-of-function mutants in PKM2-knockout cells showed that the regulatory function of PKM2 in this progress does not depend on the activity of glycolytic enzymes or protein kinases. Taken together, these findings demonstrate that PKM2 mediates the formation of the CHIP-HSP70-BAG3 protein complex and promotes the aggregation of ubiquitinated misfolded proteins, thus compensating for proteasome stress in cells., (© 2021 Federation of American Societies for Experimental Biology.)

Colorectal cancer (CRC) is the most common gastrointestinal cancer, with a high mortality rate but limited therapeutic targets. DIRAS family GTPase 2 ( DIRAS2 ) is a member of the Ras-related small G-protein family whose biological functions and underlying mechanism in CRC remain poorly understood. In this study, we identified the crucial roles of DIRAS2 in CRC. DIRAS2 expression was downregulated in CRC and closely correlated with poor prognosis. Functionally, DIRAS2 inhibited CRC cell proliferation and affected cell-cycle protein expression. Mechanistically, DIRAS2 blocked nuclear factor kappa light-chain enhancer of activated B-cell signaling pathways, inducing G0/G1 arrest. Moreover, DIRAS2 interacted with 26S proteasome non-ATPase regulatory subunit 2, which facilitates the degradation of DIRAS2 in a proteasome-mediated way. Together, these results demonstrate potential functions of DIRAS2 as a tumor-suppressor gene in CRC and reveal a distinct mechanism of DIRAS2 in CRC tumorigenesis, indicating its role as a potential biomarker and target for CRC therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The tight regulation of proteostasis is essential for physiological cellular function. Mammalian cells possess a network of mechanisms that ensure proteome integrity under normal or stress conditions. The proteasome, being the major cellular proteolytic machinery, is central to proteostasis maintenance in response to distinct intracellular and extracellular conditions. The proteasomes are multisubunit protease complexes that selectively catalyze the degradation of short-lived regulatory proteins and damaged peptides. Different forms of the proteasome complexes comprising of different subunits and attached regulators directly affect the substrate selectivity and degradation. Thus, the proteasome participates in the turnover of a multitude of factors that control key processes that affect the cellular state, such as adaptation to environmental cues, growth, development, metabolism, signaling, senescence, pluripotency, differentiation, and immunity. Aberrations on its function are related to normal processes like aging and pathological conditions such as neurodegeneration and cancer. The past few years of research have highlighted that proteasome abundance, activity, assembly, and localization are subject to a dynamic transcriptional control that secures the continuous adaptation of the proteasome to internal or external stimuli. This review focuses on the factors and signaling pathways that are involved in the regulation of the mammalian proteasome at the transcriptional level. A comprehensive understanding of proteasome regulation has critical implications on disease prevention and treatment., (© 2021 International Union of Biochemistry and Molecular Biology.)

Background: TP53 protein is lost or mutated in about half of all types of human cancers and small molecules to regulate mutant p53 repair, or interrupt ubiquitination degradation of p53 induced by E3-ubiquitin ligase Mdm2 have a potential application in clinical application. Methods: To inhibit the deubiquitinase activity of 19S proteasome and restore the p53 protein level, in this study, we utilized p53 knockout mice to test the anti-cancer effect of a specific USP14 and UCH37 inhibitor b-AP15. Results: Our results show that UCHL5, USP14 and COPS5 are upregulated in p53-related tumors, and higher expression of these genes results in a shorter overall survival in patients with p53 deficiency. Treatment with b-AP15, a UCHL5 and USP14 deubiquitinating activity inhibitor in 19S regulatory subunit, induces tumor regression and prolong the survival period of tumor-loaded mice through down-regulation of COPS5 and its downstream AP-1 and E2F1, and up-regulation of the cell cycle-related proteins p27 and Cyclin E1. Conclusions: Thus, our results suggested that inhibition of UCHL5 and USP14 deubiquitinating activity in 19S proteasome may contribute an extensive approach to preventing tumor progress due to p53 deficiency.

Apoptosis is a cell suicide process that is essential for development, tissue homeostasis and human health. Impaired apoptosis is associated with a variety of human diseases, including neurodegenerative disorders, autoimmunity and cancer. As the levels of pro- and anti-apoptotic proteins can determine the life or death of cells, tight regulation of these proteins is critical. The ubiquitin proteasome system (UPS) is essential for maintaining protein turnover, which can either trigger or inhibit apoptosis. In this review, we will describe the E3 ligases that regulate the levels of pro- and anti-apoptotic proteins and assisting proteins that regulate the levels of these E3 ligases. We will provide examples of apoptotic cell death modulations using the UPS, determined by positive and negative feedback loop reactions. Specifically, we will review how the stability of p53, Bcl-2 family members and IAPs (Inhibitor of Apoptosis proteins) are regulated upon initiation of apoptosis. As increased levels of oncogenes and decreased levels of tumor suppressor proteins can promote tumorigenesis, targeting these pathways offers opportunities to develop novel anti-cancer therapies, which act by recruiting the UPS for the effective and selective killing of cancer cells.

Background: Disruption of the blood-brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear., Methods: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen-glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test., Results: Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R., Conclusion: This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions., (© 2021. The Author(s).)

Objective: This study investigated the nature of shared transcriptomic alterations in PBMs from periodontitis and atherosclerosis to unravel molecular mechanisms underpinning their association., Methods: Gene expression data from PBMs from patients with periodontitis and those with atherosclerosis were each downloaded from the GEO database. Differentially expressed genes (DEGs) in periodontitis and atherosclerosis were identified through differential gene expression analysis. The disease-related known genes related to periodontitis and atherosclerosis each were downloaded from the DisGeNET database. A Venn diagram was constructed to identify crosstalk genes from four categories: DEGs expressed in periodontitis, periodontitis-related known genes, DEGs expressed in atherosclerosis, and atherosclerosis-related known genes. A weighted gene coexpression network analysis (WGCNA) was performed to identify significant coexpression modules, and then, coexpressed gene interaction networks belonging to each significant module were constructed to identify the core crosstalk genes., Results: Functional enrichment analysis of significant modules obtained by WGCNA analysis showed that several pathways might play the critical crosstalk role in linking both diseases, including bacterial invasion of epithelial cells, platelet activation, and Mitogen-Activated Protein Kinases (MAPK) signaling. By constructing the gene interaction network of significant modules, the core crosstalk genes in each module were identified and included: for GSE23746 dataset, RASGRP2 in the blue module and VAMP7 and SNX3 in the green module, as well as HMGB1 and SUMO1 in the turquoise module were identified; for GSE61490 dataset, SEC61G, PSMB2, SELPLG, and FIBP in the turquoise module were identified., Conclusion: Exploration of available transcriptomic datasets revealed core crosstalk genes (RASGRP2, VAMP7, SNX3, HMGB1, SUMO1, SEC61G, PSMB2, SELPLG, and FIBP) and significant pathways (bacterial invasion of epithelial cells, platelet activation, and MAPK signaling) as top candidate molecular linkage mechanisms between atherosclerosis and periodontitis., Competing Interests: The authors declare no potential conflict of interests with respect to the authorship and/or publication of this paper., (Copyright © 2021 Wanchen Ning et al.)

Autophagy plays vital roles in the interaction between the necrotrophic fungal pathogen Sclerotinia sclerotiorum and its hosts. However, so far, only little is known about the impacts of autophagy machinery in S. sclerotiorum per se on the fungal morphogenesis and pathogenesis. Here, through functional genomic approaches, we showed that SsATG8, one of the core components of the autophagy machinery, and its interactor SsNBR1, an autophagy cargo receptor, are important for vegetative growth, sclerotial formation, oxalic acid (OA) production, compound appressoria development, and virulence of S. sclerotiorum. Complementation assays with chimeric fusion constructs revealed that both LDS [AIM (ATG8 interacting motif) / LIR (LC3-interacting region) docking site] and UDS [UIM (ubiquitin-interacting motif) docking site] sites of the SsATG8 are required for its functions in autophagy and pathogenesis. Importantly, ΔSsatg8 and ΔSsnbr1 mutants showed enhanced sensitivity to the exogenous treatment with the proteasome inhibitors bortezomib and carfilzomib, and ΔSsnbr1 mutant had decreased expression of SsATG8 under the proteasomal stress conditions, suggesting that a cross-talk exists between ubiquitin-proteasome and selective autophagy pathways, which enables downstream protein degradation to proceed properly during diverse biological processes. Collectively, our data indicate that SsATG8- and SsNBR1-mediated autophagy is crucial for S. sclerotiorum development, proteasomal stress response and virulence., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The crucial roles of the long noncoding RNAs (lncRNAs) in the development of ovarian cancer (OC) have been extensively studied. According to the prediction result from the Kaplan-Meier Plotter database, high expression of lncRNA proteasome subunit α type-3 antisense RNA1 (PSMA3-AS1) is associated with the poor prognosis in patients with OC. Thus, the study aimed to investigate the role of lncRNA PSMA3-AS1 in OC. Reverse transcription quantitative polymerase chain reaction analysis revealed that PSMA3-AS1 expression was significantly upregulated in OC cells and tissues. PSMA3-AS1 silencing inhibited OC cell proliferation, migration, and invasion, as shown by results of cell counting kit-8, colony formation, wound healing, and Transwell assays, respectively. Additionally, PSMA3-AS1 deficiency suppressed tumor growth in vivo. Mechanistically, luciferase reporter and RNA pulldown assays implied that PSMA3-AS1 served as a competing endogenous RNA for miR-378a-3p to upregulate the expression of polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). GALNT3 was a target gene of miR-378a-3p in OC. Moreover, PSMA3-AS1 activated the PI3K/Akt pathway by upregulating GALNT3 expression. Overall, PSMA3-AS1 promotes OC cell proliferation, migration, invasion, and xenograft tumor growth by activating the PI3K/Akt pathway via the miR-378a-3p/GALNT3 axis., (© 2021 Wiley Periodicals LLC.)

The immunoproteasomes are specific proteasomes that clear oxidant-damaged proteins under inflammatory conditions in various diseases. Toxoplasma gondii (T. gondii) infects the central nervous system and causeencephalitis. However, the relationship between the immunoproteasomes and brain inflammation during T. gondii infection is not well characterized. In this study, we established an in vivo mouse model of T. gondii PLK strain infection via intraperitoneal injection and evaluated the expression of immunoproteasome subunits in the brains of infected mice. The results demonstrated that first, pathological changes in the brains of infected mice increase in severity over time. Second, following T. gondii infection, activated microglia and astrocytes undergo a series of functional alterations and morphological transformations, including proliferation and migration. Third, T. gondii infection induces expression of inflammatory cytokines, including IFN-γ, IL-1β, TNF-α, and IL-6. Fourth, the immunoproteasome subunits low-molecular-weight polypeptide 2 (LMP2), LMP7, and LMP10 mRNA and protein levels are significantly upregulated in T. gondii-infected mouse brains, as shown by RT-qPCR and western blot analysis, compared with that in vehicle-treated brains, and their expression is localized in the microglia, astrocytes, and neurons of T. gondii-infected brains, as determined via immunofluorescence staining. Furthermore, the western blot mean gray value for the immunoproteasome subunits and the positive microglia and astrocyte immunohistochemical signals in the brains of T. gondii-infected mice were positively correlated, indicating that the observed relationships were highly significant. Therefore, it was concluded that the induction of the immunoproteasomes is a pathogenic mechanism underlying T. gondii infection-induced inflammation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Kinesin-like calmodulin-binding protein (KCBP) is a unique kinesin with half kinesin and half myosin, with kinesin motor domain at C-terminus and myosin tail homology region 4 (MyTH4) and band 4.1, ezrin, radixin, moesin (FERM) domains at N-terminus. The special structure endows KCBP multi-intracellular functions, including cell division, trichome morphogenesis in plants, and flagellar function in algae. However, little is known about the molecular mechanism underlying these functions. Here, we identified a molecular chaperone Hsp90 as a novel binding partner with KCBP in Dunaliella salina using a yeast two-hybrid screen. Further analysis showed that Hsp90 interacted with both the N-terminal and C-terminal of DsKCBP. Since Hsp90 was involved in the stability and proteolytic turnover of numerous proteins, whether Hsp90 regulated the degradation of DsKCBP was investigated. Our results showed that both Hsp90 and DsKCBP presented in the purified proteasome, and the interaction of DsKCBP-Hsp90 was inhibited upon Hsp90 inhibitor geldanamycin treatment. The level of DsKCBP proteins was diminished remarkably indicating that the disassociation of DsKCBP from Hsp90 accelerated the degradation of the former. Furthermore, immunofluorescence results showed that the localization of DsKCBP at basal body and flagella was disappeared by Hsp90 inhibition. The increased mRNA level of DsKCBP during flagellar assembly was not obvious by geldanamycin treatment. These data provided evidence that Hsp90 protected DsKCBP from degradation by proteasome and was involved in the role of DsKCBP in flagellar assembly., (© 2021. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Previous studies have showed that proteasome activator complex subunit 2 (PSME2) may play a role in some types of cancer. However, the involvement of PSME2 in clear cell renal cell carcinoma (ccRCC) remains unknown. The aim of the present study was to assess the poorly understood function of PSME2 expression in renal carcinoma. Using bioinformatics analysis, PSME2 mRNA expression profiles were investigated, along with its potential prognostic value and its functional enrichment. Signaling pathways and putative hub genes associated with PSME2 in ccRCC were identified. Based on the bioinformatics analysis results, immunohistochemistry of human ccRCC samples and renal carcinoma cell lines (CAKI‑1 and 786‑O) transfected with short interfering RNA targeting PSME2 were analyzed using western blot analysis, reverse transcription‑quantitative PCR, immunofluorescence, and Cell Counting Kit‑8, Transwell and transmission electron microscope assays. The results showed that when PSME2 expression was knocked down, the invasive abilities of the tumor cell lines were reduced, while autophagy was enhanced. The present study demonstrated that PSME2 was associated with the invasion ability of ccRCC cell lines by inhibiting BNIP3‑mediated autophagy. In summary, PSME2 could be used as a prognostic factor and a promising therapeutic target in ccRCC.

Purpose: To assess whether contralateral parenchymal enhancement (CPE) on MRI is associated with gene expression pathways in ER+/HER2-breast cancer, and if so, whether such pathways are related to survival., Methods: Preoperative breast MRIs were analyzed of early ER+/HER2-breast cancer patients eligible for breast-conserving surgery included in a prospective observational cohort study (MARGINS). The contralateral parenchyma was segmented and CPE was calculated as the average of the top-10% delayed enhancement. Total tumor RNA sequencing was performed and gene set enrichment analysis was used to reveal gene expression pathways associated with CPE (N = 226) and related to overall survival (OS) and invasive disease-free survival (IDFS) in multivariable survival analysis. The latter was also done for the METABRIC cohort (N = 1355)., Results: CPE was most strongly correlated with proteasome pathways (normalized enrichment statistic = 2.04, false discovery rate = .11). Patients with high CPE showed lower tumor proteasome gene expression. Proteasome gene expression had a hazard ratio (HR) of 1.40 (95% CI = 0.89, 2.16; P = .143) for OS in the MARGINS cohort and 1.53 (95% CI = 1.08, 2.14; P = .017) for IDFS, in METABRIC proteasome gene expression had an HR of 1.09 (95% CI = 1.01, 1.18; P = .020) for OS and 1.10 (95% CI = 1.02, 1.18; P = .012) for IDFS., Conclusion: CPE was negatively correlated with tumor proteasome gene expression in early ER+/HER2-breast cancer patients. Low tumor proteasome gene expression was associated with improved survival in the METABRIC data., Competing Interests: Declaration of competing interest None declared., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The complexity of breast cancer includes many interacting biological processes that make it difficult to find appropriate therapeutic treatments. Therefore, identifying potential diagnostic and prognostic biomarkers is urgently needed. Previous studies demonstrated that 26S proteasome delta subunit, non-ATPase (PSMD) family members significantly contribute to the degradation of damaged, misfolded, abnormal, and foreign proteins. However, transcriptional expressions of PSMD family genes in breast cancer still remain largely unexplored. Consequently, we used a holistic bioinformatics approach to explore PSMD genes involved in breast cancer patients by integrating several high-throughput databases, including The Cancer Genome Atlas (TCGA), cBioPortal, Oncomine, and Kaplan-Meier plotter. These data demonstrated that PSMD1, PSMD2, PSMD3, PSMD7, PSMD10, PSMD12, and PSMD14 were expressed at significantly higher levels in breast cancer tissue compared to normal tissues. Notably, the increased expressions of PSMD family genes were correlated with poor prognoses of breast cancer patients, which suggests their roles in tumorigenesis. Meanwhile, network and pathway analyses also indicated that PSMD family genes were positively correlated with ubiquinone metabolism, immune system, and cell-cycle regulatory pathways. Collectively, this study revealed that PSMD family members are potential prognostic biomarkers for breast cancer progression and possible promising clinical therapeutic targets.

Impaired proteasome activity due to genetic variants of certain subunits might lead to proteasome-associated autoinflammatory syndromes (PRAAS). Here we report a de novo heterozygous missense variant of the PSMB9 proteasome subunit gene in two unrelated Japanese infants resulting in amino acid substitution of the glycine (G) by aspartic acid (D) at position 156 of the encoded protein β1i. In addition to PRAAS-like manifestations, these individuals suffer from pulmonary hypertension and immunodeficiency, which are distinct from typical PRAAS symptoms. The missense variant results in impaired immunoproteasome maturation and activity, yet ubiquitin accumulation is hardly detectable in the patients. A mouse model of the heterozygous human genetic variant (Psmb9 G156D/+ ) recapitulates the proteasome defects and the immunodeficiency phenotype of patients. Structurally, PSMB9 G156D interferes with the β-ring-βring interaction of the wild type protein that is necessary for 20S proteasome formation. We propose the term, proteasome-associated autoinflammatory syndrome with immunodeficiency (PRAAS-ID), to indicate a separate category of autoinflammatory diseases, similar to, but distinct from PRAAS, that describes the patients in this study., (© 2021. The Author(s).)

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB., Competing Interests: AS, ZH, DA, JD, AD, JC, SM, CY, LH, DF, RC, TY No competing interests declared, CH Reviewing editor, eLife, (© 2021, Song et al.)

During embryonic development in mammals, the testicles generally descend into the scrotum, making the testicular temperature 2-4 °C lower than the core temperature via heat exchange and clearance, and thus more beneficial for normal spermatogenesis. Failure to descend, known as cryptorchidism, carries a series of risks such as infertility and testicular cancer. However, some mammals have evolved abdominal testes while maintaining healthy reproduction. To explore the underlying molecular mechanism, we conducted comparative genomic analyses and functional assays on the spermatogenesis-related ubiquitin-proteasome system (UPS) genes essential to sperm formation in representative laurasiatherians. Here, positive selection and rapid evolution of spermatogenesis-related UPS genes were identified in the abdominal testicular laurasiatherians. Moreover, potential convergent amino acids were found between distantly related species with similar abdominal testicles and functional analyses showed RNF8 (V437I) in abdominal testicular species (437I) has a stronger ubiquitination ability, which suggests that the mammals with abdominal testes might exhibit enhanced sperm cell histone clearance to maintain sperm formation. This evidence implies that, in response to "cryptorchidism injury", spermatogenesis-related UPS genes in the abdominal testicular species might have undergone adaptive evolution to stabilize sperm formation. Thus, our study could provide some novel insights into the reproductive adaptation in abdominal testicular mammals.

Reversible phosphorylation has emerged as an important mechanism for regulating proteasome function in various physiological processes. Essentially all proteasome phosphorylations characterized thus far occur on proteasome holoenzyme or subcomplexes to regulate substrate degradation. Here, we report a highly conserved phosphorylation that only exists on the unassembled α5 subunit of the proteasome. The modified residue, α5-Ser16, is within a SP motif typically recognized by cyclin-dependent kinases (CDKs). Using a phospho-specific antibody generated against this site, we found that α5-S16 phosphorylation is mitosis-specific in both yeast and mammalian cells. Blocking this site with a S16A mutation caused growth defect and G2/M arrest of the cell cycle. α5-S16 phosphorylation depends on CDK1 activity and is highly abundant in some but not all mitotic cells. Immunoprecipitation and mass spectrometry (IP-MS) studies identified numerous proteins that could interact with phosphorylated α5, including PLK1, a key regulator of mitosis. α5-PLK1 interaction increased upon mitosis and could be facilitated by S16 phosphorylation. CDK1 activation downstream of PLK1 activity was delayed in S16A mutant cells, suggesting an important role of α5-S16 phosphorylation in regulating PLK1 and mitosis. These data have revealed an unappreciated function of "exo-proteasome" phosphorylation of a proteasome subunit and may bring new insights to our understanding of cell cycle control.

Lipodystrophic syndromes (LS) constitute a clinically and genetically heterogeneous group of diseases characterized by a loss of adipose tissue. These syndromes are usually associated with metabolic complications, which are determinant for morbidity and mortality. The classical forms of LS include partial, generalized, and progeroid lipodystrophies. They are usually due to defects in proteins playing a key role in adipogenesis and adipocyte functions. More recently, systemic disorders combining lipodystrophy and multiple organ dysfunction have been described, including autoinflammatory syndromes, mitochondrial disorders, as well as other complex entities. To date, more than thirty genes have been implicated in the monogenic forms of LS, but the majority of them remain genetically-unexplained. The associated pathophysiological mechanisms also remain to be clarified in many instances. Next generation sequencing-based approaches allow simultaneous testing of multiple genes and have become crucial to speed up the identification of new disease-causing genes. The challenge for geneticists is now the interpretation of the amount of available genetic data, generated especially by exome and whole-genome sequencing. International recommendations on the interpretation and classification of variants have been set up and are regularly reassessed. Very close collaboration between geneticists, clinicians, and researchers will be necessary to make rapid progress in understanding the molecular and cellular basis of these diseases, and to promote personalized medicine., Competing Interests: Disclosure of interest The author declares that she has no conflict of interest concerning this article, (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Ginsenoside 20(S)-Rg3 is identified as an active saponin monomer which derived from red ginseng and is demonstrated to play an anti-tumor role in diverse cancers. MicroRNAs (miRNAs) are important regulators in the progression of cancers, containing esophageal squamous cell carcinoma (ESCC). It was reported that microRNA 324-5p (miR-324-5p) exerted critical functions in some cancers; however, the detailed molecular mechanism of miR-324-5p mediated by 20(S)-Rg3 to suppress cell viability in ESCC has not been explored. Herein, we explored the function of 20(S)-Rg3 or miR-324-5p on ESCC cell viability by MTT assay, colony formation assay, flow cytometry analysis and western blot analysis. The binding of miR-324-5p to its target gene, proteasome activator subunit 3 (PSME3), was confirmed through RNA pull down and luciferase reporter assays. The results indicated that 20(S)-Rg3 significantly inhibited cell viability and the cell cycle and facilitated cell apoptosis. Furthermore, this effect was strengthened with the increased concentration of 20(S)-Rg3. Moreover, we found that miR-324-5p level was increased under 20(S)-Rg3 treatment. Additionally, overexpressed miR-324-5p inhibited ESCC cell viability, and downregulated miR-324-5p recovered inhibited cell viability caused by 20(S)-Rg3. Further exploration verified that miR-324-5p targeted PSME3, and PSME3 deficiency countervailed the effect of miR-324-5p inhibition on ESCC cell viability under 20(S)-Rg3 treatment. Conclusively, 20(S)-Rg3 suppresses cell viability in ESCC via mediating miR-324-5p-targeted PSME3.

Background: Protein degradation systems play crucial roles in all the kingdoms of life. Their natural function is to eliminate proteins that are improperly synthesized, damaged, aggregated, or short-lived, ensuring the timely and accurate regulation of the response to abrupt environmental changes. Thus, proteolysis plays an important role in protein homeostasis, quality control, and the control of regulatory processes, such as adaptation and cell development. Except for the lysosome, ATPases Associated with various cellular Activities (AAA+) ATPase-protease complex is another major protein degradation system in the cell., Methods and Results: The AAA+ ATPase-protease complex is a giant energy-dependent protease complex found in almost all kinds of cells, including bacteria, archaea and eukarya. Based on sequence analysis of ClpQ (HslV) and 20S proteasome beta subunits, it was found that bacterial ClpQ possess multiple same highly conserved motifs with 20S proteasome beta subunits of archaea and eukaryote. In this review, we also discussed the structure and functional mechanism, protein degradation signals and pathogenic role of proteasome / Clp protease complex in prokaryotes., Conclusion: Bacterial protein degradation systems play important roles in stress tolerance, protein quality control, DNA protection, transcription and pathogenicity of bacteria. But our current knowledge of the bacterial protease system is incomplete, and further research into the Clp protease complex and associated protein degradation signals will extend our understanding of the metabolism, physiology, reproduction, and pathogenicity of bacteria., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Previous studies suggested that cancer cells resemble neural stem/progenitor cells in regulatory network, tumorigenicity, and differentiation potential, and that neural stemness might represent the ground or basal state of differentiation and tumorigenicity. The neural ground state is reflected in the upregulation and enrichment of basic cell machineries and developmental programs, such as cell cycle, ribosomes, proteasomes, and epigenetic factors, in cancers and in embryonic neural or neural stem cells. However, how these machineries are concertedly regulated is unclear. Here, we show that loss of neural stemness in cancer or neural stem cells via muscle-like differentiation or neuronal differentiation, respectively, caused downregulation of ribosome and proteasome components and major epigenetic factors, including PRMT1, EZH2, and LSD1. Furthermore, inhibition of PRMT1, an oncoprotein that is enriched in neural cells during embryogenesis, caused neuronal-like differentiation, downregulation of a similar set of proteins downregulated by differentiation, and alteration of subcellular distribution of ribosome and proteasome components. By contrast, PRMT1 overexpression led to an upregulation of these proteins. PRMT1 interacted with these components and protected them from degradation via recruitment of the deubiquitinase USP7, also known to promote cancer and enriched in embryonic neural cells, thereby maintaining a high level of epigenetic factors that maintain neural stemness, such as EZH2 and LSD1. Taken together, our data indicate that PRMT1 inhibition resulted in repression of cell tumorigenicity. We conclude that PRMT1 coordinates ribosome and proteasome activity to match the needs for high production and homeostasis of proteins that maintain stemness in cancer and neural stem cells., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often considered un-druggable, recent work has demonstrated successful targeting of MYB by low molecular weight compounds. This has fueled the notion that inhibition of MYB has potential as a therapeutic approach against MYB-driven malignancies. Here, we have used a MYB reporter cell line to screen a library of FDA-approved drugs for novel MYB inhibitors. We demonstrate that proteasome inhibitors have significant MYB-inhibitory activity, prompting us to characterize the proteasome inhibitor oprozomib in more detail. Oprozomib was shown to interfere with the ability of the co-activator p300 to stimulate MYB activity and to exert anti-proliferative effects on human AML and ACC cells. Overall, our work demonstrated suppression of oncogenic MYB activity as a novel result of proteasome inhibition., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Persistent coinfection with Helicobacter pylori and Epstein-Barr virus (EBV) promotes aggressive gastric carcinoma (GC). The molecular mechanisms underlying the aggressiveness in H. pylori and EBV-mediated GC are not well characterized. We investigated the molecular mechanism involved in H. pylori- and EBV-driven proliferation of gastric epithelial cells. Results showed that the coinfection is significantly more advantageous to the pathogens as coinfection creates a microenvironment favorable to higher pathogen-associated gene expression. The EBV latent genes ebna1 and ebna3c are highly expressed in the coinfection compared to lone EBV infection at 12 and 24 h. The H. pylori-associated genes 16S rRNA, cagA , and babA were also highly expressed during coinfection compared to H. pylori alone. In addition, upregulation of gankyrin, which is a small oncoprotein, modulates various cell signaling pathways, leading to oncogenesis. Notably, the knockdown of gankyrin decreased the cancer properties of gastric epithelial cells. Gankyrin showed a similar expression pattern as that of ebna3c at both transcript and protein levels, suggesting a possible correlation. Further, EBV and H. pylori created a microenvironment that induced cell transformation and oncogenesis through dysregulation of the cell cycle regulatory ( ccnd1 , dapk3 , pcna , and akt ), GC marker ( abl1 , tff-2 , and cdx2 ), cell migration ( mmp3 and mmp7 ), DNA response ( pRB , pten , and p53 ), and antiapoptotic ( bcl2 ) genes in infected gastric epithelial cells through gankyrin. Our study provides a new insight into the interplay of two oncogenic agents (H. pylori and EBV) that leads to an enhanced carcinogenic activity in gastric epithelial cells through overexpression of gankyrin. IMPORTANCE In the present study, we evaluated the synergistic effects of EBV and H. pylori infection on gastric epithelial cells in various coinfection models. These coinfection models were among the first to depict the exposures of gastric epithelial cells to EBV followed by H. pylori; however, coinfection models exist that narrated the scenario upon exposure to H. pylori followed by that to EBV. We determined that a coinfection by EBV and H. pylori enhanced the expression of oncogenic protein gankyrin. The interplay between EBV and H. pylori promoted the oncogenic properties of AGS cells like elevated focus formation, cell migration, and cell proliferation through gankyrin. EBV and H. pylori mediated an enhanced expression of gankyrin, which further dysregulated cancer-associated genes such as cell migratory, tumor suppressor, DNA damage response, and proapoptotic genes.

The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

Identification of proteasomal spliced peptides (PSPs) by mass spectrometry (MS) is not possible with traditional search engines. Here, we provide a protocol for running RHybridFinder (RHF), an R package for the computational inference of putative PSPs detected by MS. RHF extracts high confidence scored de novo sequenced peptides identified by PEAKS software. Those peptides are then matched to protein databases to infer cis- or trans-spliced major histocompatibility complex (MHC)-associated peptides. RHF is relatively fast and straightforward. PSPs have to be validated experimentally. For complete details on the use and execution of the original protocol, please refer to Faridi et al. (2018)., Competing Interests: The authors declare no competing interests., (Crown Copyright © 2021.)

Rhodopsin ( RHO ) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHO P23H , results in its misfolding and retention in the endoplasmic reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD). Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins, such as RHO P23H , includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHO P23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.

Recently some evidence has demonstrated the significance of PSMB8 in various malignancies. Nevertheless, PSMB8 (proteasome subunit beta 8), more familiar in the field of immunology contributing to the process of antigen presentation, is indeterminate in the role as a survival predictor of human pan-cancer. Besides, how PSMB8 interacts with immune cell infiltration in the tumor microenvironment requires further research. We then penetrated into the analysis of the PSMB8 expression profile among 33 types of cancer in the TCGA database. The results show that overexpression of PSMB8 was associated with poor clinical outcomes in overall survival (Sartorius et al. in Oncogene 35(22):2881-2892, 2016), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI) in most cancer varieties. In addition, there existed distinctly positive correlations between PSMB8 and immunity, reflected straightforwardly in the form of immune scores, tumor-infiltrating immune cells (TIICs) abundance, microsatellite instability, tumor mutation burden, and neoantigen level. Notably, specific markers of dendrite cells exhibited the tightest association with PSMB8 expression in terms of tumor-related immune infiltration patterns. Moreover, gene enrichment analysis showed that elevated PSMB8 expression was related to multiple immune-related pathways. We finally validated the PSMB8 expression in our local breast samples via quantitative PCR assays and concluded that PSMB8 appeared to perform well in predicting the survival outcome of BRCA patients. These findings elucidate the pivotal role of the antigen presentation-related gene PSMB8, which could potentially serve as a robust biomarker for prognosis determination in multiple cancers., (© 2021. The Author(s).)

Background: Tuberculosis (TB) is a serious chronic bacterial infection caused by Mycobacterium tuberculosis (MTB). It is one of the deadliest diseases in the world and a heavy burden for people all over the world. However, the hub genes involved in the host response remain largely unclear., Methods: The data set GSE11199 was studied to clarify the potential gene network and signal transduction pathway in TB. The subjects were divided into latent tuberculosis and pulmonary tuberculosis, and the distribution of differentially expressed genes (DEGs) was analyzed between them using GEO2R. We verified the enriched process and pathway of DEGs by making use of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). The construction of protein-protein interaction (PPI) network of DEGs was achieved through making use of the Search Tool for the Retrieval of Interacting Genes (STRING), aiming at identifying hub genes. Then, the hub gene expression level in latent and pulmonary tuberculosis was verified by a boxplot. Finally, through making use of Gene Set Enrichment Analysis (GSEA), we further analyzed the pathways related to DEGs in the data set GSE11199 to show the changing pattern between latent and pulmonary tuberculosis., Results: We identified 98 DEGs in total in the data set GSE11199, 91 genes upregulated and 7 genes downregulated included. The enrichment of GO and KEGG pathways demonstrated that upregulated DEGs were mainly abundant in cytokine-mediated signaling pathway, response to interferon-gamma, endoplasmic reticulum lumen, beta-galactosidase activity, measles, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, etc. Based on the PPI network, we obtained 4 hub genes with a higher degree, namely, CTLA4, GZMB, GZMA, and PRF1. The box plot showed that these 4 hub gene expression levels in the pulmonary tuberculosis group were higher than those in the latent group. Finally, through Gene Set Enrichment Analysis (GSEA), it was concluded that DEGs were largely associated with proteasome and primary immunodeficiency., Conclusions: This study reveals the coordination of pathogenic genes during TB infection and offers the diagnosis of TB a promising genome. These hub genes also provide new directions for the development of latent molecular targets for TB treatment., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Tiancheng Zhang et al.)

Proteasome assembly utilizes multiple dedicated assembly chaperones and is regulated by signaling pathways that respond to diverse stress conditions. To discover new factors influencing proteasome base assembly, we screened a tiled high-copy yeast genomic library to identify dosage suppressors of a temperature-sensitive proteasome regulatory particle (RP) base mutant. The screen identified negative salt tolerance 1 (Nst1), a protein that when overexpressed specifically suppressed the temperature sensitivity and proteasome-assembly defects of multiple base mutants. Nst1 overexpression reduced cytosolic RP ATPase (Rpt) aggregates in nas6 Δ rpn14 Δ cells, which lack two RP assembly chaperones. Nst1 is highly polar and predicted to have numerous intrinsically disordered regions, characteristics commonly found in proteins that can segregate into membraneless condensates. In agreement with this, both endogenous and overexpressed Nst1 could form cytosolic puncta that colocalized with processing body (P-body) components. Consistent with the accumulation of translationally inactive mRNAs in P-bodies, Nst1 overexpression inhibited global protein translation in nas6 Δ rpn14 Δ cells. Translational inhibition is known to suppress aggregation and proteasome assembly defects in base mutants under heat stress . Our data indicate that Nst1 is a previously overlooked P-body component that, when expressed at elevated levels inhibits translation, prevents Rpt subunit aggregation and rescues proteasome assembly under stress conditions.

Maintaining the integrity of organelles despite the cellular disturbances that arise during stress is essential for life. To ensure organelle proteostasis (protein homeostasis), plants have evolved multitiered quality control mechanisms that work together to repair or recycle the damaged organelles. Despite recent advances, our understanding of plant organelle quality control mechanisms is far from complete. Especially, the crosstalk between different quality control pathways remains elusive. Here, we highlight recent advances on organelle quality control, focusing on the targeted protein degradation pathways that maintain the homeostasis of the endoplasmic reticulum (ER), chloroplast, and mitochondria. We discuss how plant cells decide to employ different degradation pathways and propose tools that could be used to discover the missing components in organelle quality control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

DNA-binding proteins, including transcription factors (TFs), play essential roles in various cellular processes and pathogenesis of diseases, deeming to be potential therapeutic targets. However, these proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand-binding pocket. Proteolysis-targeting chimera (PROTAC) technology has been developed by engineering a bifunctional molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through the proteasome pathway. Here, the development of oligonucleotide-based PROTAC (O'PROTACs), a class of noncanonical PROTACs in which a TF-recognizing double-stranded oligonucleotide is incorporated as a binding moiety of POI is reported. It is demonstrated that O'PROTACs of lymphoid enhancer-binding factor 1 (LEF1) and ETS-related gene (ERG), two highly cancer-related transcription factors, successfully promote degradation of these proteins, impede their transcriptional activity, and inhibit cancer cell growth in vitro and in vivo. The programmable nature of O'PROTACs indicates that this approach is also applicable to destruct other TFs. O'PROTACs not only can serve as a research tool but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Arsenic is considered a worldwide pollutant that can be present in drinking water. Arsenic exposure is associated with various diseases, including cancer. Antioxidants as selenite and α-tocopherol-succinate have been shown to modulate arsenic toxic effects. Since changes in STAT3 and PSMD10 gene expression have been associated with carcinogenesis, the aim of this study was to evaluate the effect of arsenic exposure and co-treatments with selenite or α-tocopherol-succinate on the expression of these genes, in the livers of chronically exposed Syrian golden hamsters. Animals were divided into six groups: (i) control, (ii) chronically treated with 100 ppm arsenic, (iii) treated with 6 ppm α-tocopherol-succinate (α-TOS), (iv) treated with 8.5 ppm selenite, (v) treated with arsenic + α-TOS, and (vi) treated with arsenic + selenite. Urine samples and livers were collected after 20 weeks of continuous exposure. The urine samples were analyzed for arsenic species by atomic absorption spectrophotometry, and real-time RT-qPCR analysis was performed for gene expression evaluation. A reduction in STAT3 expression was observed in the selenite-treated group. No differences in PSMD10 expression were found among groups. Histopathological analysis revealed hepatic lymphocytosis in selenite-treated animals. As a conclusion, long-term exposure to arsenic does not significantly alter the expression of STAT3 and PSMD10 oncogenes in the livers of hamsters; however, selenite down-regulates STAT3 expression and provokes lymphocytosis.

Ever since the ubiquitin proteasome system was characterized, efforts have been made to manipulate its function to abrogate the progression of cancer. As a result, the anti-cancer drugs bortezomib, carfilzomib, and ixazomib targeting the 26S proteasome were developed to treat multiple myeloma, mantle cell lymphoma, and diffuse large B-cell lymphoma, among others. Despite success, adverse side effects and drug resistance are prominent, raising the need for alternative therapeutic options. We recently demonstrated that knockdown of the 19S regulatory components, 26S proteasome non-ATPase subunits 1 ( PSMD1 ) and 3 ( PSMD3 ), resulted in increased apoptosis of chronic myeloid leukemia (CML) cells, but had no effect on normal controls, suggesting they may be good targets for therapy. Therefore, we hypothesized that PSMD1 and PSMD3 are potential targets for anti-cancer therapeutics and that their relevance stretches beyond CML to other types of cancers. In the present study, we analyzed PSMD1 and PSMD3 mRNA and protein expression in cancerous tissue versus normal controls using data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), comparing expression with overall survival. Altogether, our data suggest that PSMD1 and PSMD3 may be novel putative targets for cancer prognosis and therapy that are worthy of future investigation.

Cholangiocarcinoma (CCA) has been well known as the second most common primary tumor of hepatobiliary system. PSMC2 (proteasome 26S subunit ATPase 2) is a key member of the 19S regulatory subunit of 26S proteasome, responsible for catalyzing the unfolding and translocation of substrates into the 20S proteasome, whose role in CCA is totally unknown. In this study, the results of immunohistochemistry analysis showed the upregulation of PSMC2 in CCA tissues compared with normal tissues, which was statistically analyzed to be associated with CCA tumor grade. Subsequently, the loss-of-function study suggested that knockdown of PSMC2 significantly suppressed cell proliferation, cell migration, promoted cell apoptosis and arrested cell cycle distribution in vitro . The decreased tumorigenicity of CCA cells with PSMC2 knockdown was confirmed in vivo by using mice xenograft model. In PSMC2 knockdown cells, pro-apoptotic protein Caspase3 was upregulated; anti-apoptotic proteins such as Bcl-2 and IGF-II were downregulated; among EMT markers, E-cadherin was upregulated while N-cadherin and Vimentin were downregulated, by which may PSMC2 regulates cell apoptosis and migration. Furthermore, through RNA-seq and verification by qPCR, western blotting and co-IP assays, CDK1 was identified as the potential downstream of PSMC2 mediated regulation of CCA. PSMC2 and CDK1 showed mutual regulation effects on expression level of each other. Knockdown of PSMC2 could aggregate the influence of CDK1 knockdown on cellular functions of CCA cells. In summary, our findings suggested that PSMC2 possesses oncogene-like functions in the development and progression of CCA through regulating CDK1, which may be used as an effective therapeutic target in CCA treatment.

The cyanobacterial non-protein amino acid α-amino-β-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system x c &#95; and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)