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The review is focused on recent drug discovery advances based on targeted protein degradation strategies. This new area of research has exploded leading to the development of potential drugs useful in a large variety of human diseases. They first target disease relevant proteins difficult to counteract with other classical strategies and extend now to aggregates, organelles, nucleic acids or lipidic droplets. These degraders engaged either the ubiquitin-proteasome system for PROTACs and molecular glues (first generation), or the lysosomal system via endosome-lysosome degradation (LYTACs) and autophagy-lysosome degradation (ATTEC, AUTAC, AUTOTAC) (following generations of degraders). PROTACs have expanded from the orthodox heterobifunctional ones to new derivatives such as homo-PROTACs, pro-PROTACs, CLIPTACs, HaloPROTACs, PHOTOTACs, Bac-PROTACs, AbTACs, ARN-PROTACs. The small molecular-weight molecular glues induce the formation of new ternary complexes which implicate the targeted protein and an ubiquitin ligase E3 allowing the protein ubiquinitation followed by its proteasomal degradation. Lysosomal degraders (LYTAC, ATTEC, AUTAC, AUTOTAC) specifically recognize extracellular and membrane proteins or dysfunctional organelles and transport them into lysosomes where they are degraded. They overcome the limitations observed with proteasomal degradations induced by PROTAC and molecular glues and demonstrate their potential to treat human diseases, especially neurodegenerative ones. Pharmaceutical companies are engaged at the world level to develop these new potential drugs targeting cancers, immuno-inflammatory and neurodegenerative diseases as well as a variety of other ones. Efficiency and risks for these novel therapeutic strategies are discussed., (© Société de Biologie, 2024.)

Background and Aims: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs)., Methods: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers., Results and Conclusions: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes., (© 2021 Research Society on Alcoholism.)

The supply of oven-fresh bakery products to consumers has been improved by frozen dough technology; however, freeze-thaw stress decreases the activity of yeast cells. To breed better baker's yeasts for frozen dough, it is important to understand the factors affecting freeze-thaw stress tolerance in baker's yeast. We analyzed the stress response in IB1411, a spore clone from Saccharomyces cerevisiae Shirakami kodama yeast, with an exceptionally high tolerance to freeze-thaw stress. Genes encoding trehalose-6-phosphate synthase (TPS1), catalase (CTT1), and disaggregase (HSP104) were highly expressed in IB1411 cells even under conditions of non-stress. The expression of Hsp104 protein was also higher in IB1411 cells even under non-stress conditions. Deletion of HSP104 (hsp104Δ) in IB1411 cells reduced the activity of the ubiquitin-proteasome system (UPS). By monitoring the accumulation of aggregated proteins using the ΔssCPY*-GFP fusion protein under freeze-thaw stress or treatment with proteasomal inhibitor, we found that IB1411 cells resolved aggregated proteins faster than the hsp104Δ strain. Thus, Hsp104 seems to contribute to freeze-thaw tolerance by maintaining UPS activity via the disaggregation of aggregated proteins. Lastly, we found that the IB1411 cells maintained high leavening ability in frozen dough as compared with the parental strain, Shirakami kodama yeast, and thus will be useful for making bread.

Immunodominance is a complex and highly debated topic of T cell biology. The current SARS-CoV-2 pandemic has provided the opportunity to profile adaptive immune responses and determine molecular factors contributing to emerging responses towards immunodominant viral epitopes. Here, we discuss parameters that alter the dynamics of CD8 viral epitope processing, generation and T-cell responses, and how immunodominance counteracts viral immune escape mechanisms that develop in the context of emerging SARS-CoV-2 variants., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.

Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Transcription-translation coupling leads to the production of proteins that are key for controlling essential neuronal processes that include neuronal development and changes in synaptic strength. Although these events have been a prevailing theme in neuroscience, the regulation of proteins via posttranslational signaling pathways are equally relevant for these neuronal processes. Ubiquitin is one type of posttranslational modification that covalently attaches to its targets/substrates. Ubiquitination of proteins play a key role in multiple signaling pathways, the predominant being removal of its substrates by a large molecular machine called the proteasome. Here, I review 40 years of progress on ubiquitination in the nervous system at glutamatergic synapses focusing on axon pathfinding, synapse formation, presynaptic release, dendritic spine formation, and regulation of postsynaptic glutamate receptors. Finally, I elucidate emerging themes in ubiquitin biology that may challenge our current understanding of ubiquitin signaling in the nervous system., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: MiR-466 has been reported to exert a tumor-suppressive role in several cancers, including colorectal cancer and osteosarcoma, but its clinical significance and functional mechanisms in breast cancer (BC) pathogenesis still remain elusive., Patients and Methods: The expression of miR-466 was determined using reverse transcription quantitative PCR. The clinical significance of miR-466 in BC patients was assessed by Chi-square test, Kaplan-Meier method and Cox regression analyses. Functional experiments, including CCK-8 and transwell assays, were performed to analyze cell proliferation, migration and invasion ability. The association between miR-466 and proteasome subunit α7 (PSMA7) was confirmed by Luciferase reporter assay., Results: Here, we first observed that the expression of miR-466 was significantly downregulated in BC tissues and cell lines. The decreased miR-466 expression was significantly associated with tumor size (p = 0.003), lymph node metastasis (p = 0.008), TNM stage (p = 0.032) and poor survival rate. In addition, miR-466 was identified as an independent prognostic factor for BC patients. We further found that the overexpression of miR-466 significantly inhibited cell proliferation, migration and invasion. Mechanistically, PSMA7 was a potential target gene of miR-466 and negatively regulated miR-466 in BC cells. Oncomine database and Kaplan-Meier overall survival analysis indicated that upregulation of PSMA7 was associated with poor prognosis of BC patients. The rescue experiments demonstrated that PSMA7 overexpression reversed the effects of miR-466 on cell proliferation, migration, invasion and EMT transcription factors (E-cadherin, N-cadherin, and vimentin)., Conclusions: Collectively, these results suggest that the miR-466/PSMA7 axis might have potential as a therapeutic target for BC treatment.

Myocardial ischemia/reperfusion (I/R) injury induces cardiomyocyte apoptosis to deteriorate heart function. Thus, how to inhibit cardiomyocyte apoptosis is the focus of recent researches. Proteasome family member PSMB4 (proteasome subunit beta type-4) promotes cell survival. The relationship between PSMB4 and cardiomyocyte apoptosis during myocardial I/R is unknown. In this study, PSMB4 expression increased in rat myocardial I/R model, positively correlated with cleaved caspase-3 expression, negatively correlated with Bcl-2 expression. In vitro, neonatal ventricle cardiomyocyte hypoxia/reoxygenation (H/R) model was constructed to mimic myocardial I/R. PSMB4 silence promoted cardiomyocyte apoptosis and IκBα expression, inhibited the activation of NF-κB. On the contrary, PSMB4 overexpession inhibited cardiomyocyte apoptosis and IκBα expression, promoted the activation of NF-κB. Additionally, PSMB4-IκBα interaction was identified, suggesting that PSMB4 might participate in the proteasome dependent degradation of IκBα. The data indicates that PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial I/R, which can supply novel molecular target for the treatment of ischemic heart disease., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Chemical knockdown of therapeutic targets using proteolysis targeting chimeras (PROTACs) is a rapidly developing field in drug discovery, but PROTACs are bifunctional molecules that generally show poor bioavailability due to their relatively high molecular weight. Recent developments aimed at the development of next-generation PROTACs include the in vivo synthesis of PROTAC molecules, and the exploitation of PROTACs as chemical tools for in vivo synthesis of ubiquitinated proteins. This short review covers recent advances in these areas and discusses the prospects for in vivo synthetic PROTAC technology., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD's helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign - DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Neurodegeneration in the central nervous system (CNS) is a defining feature of organismal aging that is influenced by peripheral tissues. Clinical observations indicate that skeletal muscle influences CNS aging, but the underlying muscle-to-brain signaling remains unexplored. In Drosophila, we find that moderate perturbation of the proteasome in skeletal muscle induces compensatory preservation of CNS proteostasis during aging. Such long-range stress signaling depends on muscle-secreted Amyrel amylase. Mimicking stress-induced Amyrel upregulation in muscle reduces age-related accumulation of poly-ubiquitinated proteins in the brain and retina via chaperones. Preservation of proteostasis stems from the disaccharide maltose, which is produced via Amyrel amylase activity. Correspondingly, RNAi for SLC45 maltose transporters reduces expression of Amyrel-induced chaperones and worsens brain proteostasis during aging. Moreover, maltose preserves proteostasis and neuronal activity in human brain organoids challenged by thermal stress. Thus, proteasome stress in skeletal muscle hinders retinal and brain aging by mounting an adaptive response via amylase/maltose., Competing Interests: Declaration of interests M.R. and F.D. are named co-inventors of a pending U.S. provisional patent application based in part on the research reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

C9orf72 hexanucleotide repeat expansion (HRE) is the major genetic cause underpinning frontotemporal lobar degeneration (FLTD) and amyotrophic lateral sclerosis (ALS). C9orf72 HRE-associated pathogenesis involves both loss-of-function, through reduced C9orf72 levels, and gain-of-function mechanisms, including formation of RNA foci and generation of dipeptide repeat (DPR) proteins. In addition, dysfunctional protein degradation pathways, i.e. autophagy and ubiquitin-proteasome system (UPS), are suggested. Our aim was to study the gain-of-function mechanisms in the context of the function of protein degradation pathways as well as the regulation of the DPR proteins through these pathways. To this end, we expressed the pathological HRE in neuronal N2a cells and mouse primary cortical neurons. Protein degradation pathways were modulated to induce or block autophagy or to inhibit UPS. In addition, proteasomal activity was assessed. The C9orf72 HRE-expressing N2a cells and neurons were confirmed to produce RNA foci and DPR proteins, predominantly the Poly-GP proteins. However, the presence of these pathological hallmarks did not result in alterations in autophagy or proteasomal activity in either of the studied cell types. In N2a cells, Poly-GP proteins appeared in soluble forms and Lactacystin-mediated UPS inhibition increased their levels, indicating proteasomal regulation. Similar effects were not observed in cortical neurons, where the Poly-GP proteins formed also higher molecular weight forms. These results suggest a cell type-specific morphology and regulation of the DPR proteins. Further studies in other model systems may shed additional light onto the effects of the C9orf72 HRE on cellular protein degradation pathways and the regulation of the DPR protein levels., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

In mammals, the oviduct retains sperm, forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and secondly, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and channeled with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein, we found that the release of sperm from an immobilized oviduct glycan, a six-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis-X trisaccharide. Thus, CatSper may be required for release of sperm from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release requires both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

The thymus provides a microenvironment that supports the generation and selection of T cells. Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) are essential components of the thymic microenvironment and present MHC-associated self-antigens to developing thymocytes for the generation of immunocompetent and self-tolerant T cells. Proteasomes are multicomponent protease complexes that degrade ubiquitinated proteins and produce peptides that are destined to be associated with MHC class I molecules. cTECs specifically express thymoproteasomes that are essential for optimal positive selection of CD8 + T cells, whereas mTECs, which contribute to the establishment of self-tolerance in T cells, express immunoproteasomes. Immunoproteasomes are also detectable in dendritic cells and developing thymocytes, additionally contributing to T cell development in the thymus. In this review, we summarize the functions of proteasomes expressed in the thymus, focusing on recent findings pertaining to the functions of the thymoproteasomes and the immunoproteasomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Frantzeskakis, Takahama and Ohigashi.)

Accumulation of phosphorylated tau (p-tau) has long been an underappreciated hallmark of Alzheimer's disease. Tau is one of the major components of microtubule networks in neurons, and its abnormal phosphorylation and aggregation are closely related to the impairment of axonal transport. Abnormalities in axonal transport can impede autophagy in neurons, interrupting the autophagic clearance of amyloid beta. The ubiquitin proteasome system (UPS) maintains intracellular proteostasis by degrading abnormal or redundant proteins. Ever-mounting evidence suggests that UPS deficits contribute to p-tau accumulation. And targeting UPS attenuates tau pathology. This review endeavors to exam the potential role of UPS in p-tau aggregation, and how pathogenic tau may inflict other abnormalities such as amyloid beta accumulation in Alzheimer's disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Atherosclerosis is a chronic inflammatory disease of the arterial wall. It has been known that development of atherosclerosis is closely related to activation of tumor necrosis factor α (TNF-α). The objective of this study was to elucidate the effects of TNF-α blockade with brusatol on endothelial activation under pro-atherosclerotic conditions. To this end, we examined the effects of brusatol on TNF-α-induced intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in human aortic endothelial cells (HAECs) using western blot and THP-1 adhesion assays. Brusatol induced a decrease in TNF-α-induced ICAM-1 and VCAM-1 expression through inhibiting TNFR1 expression, leading to suppression of endothelial inflammation independently of the NRF2 (nuclear factor erythroid 2-related factor 2) pathway. The mechanism underlying brusatol-induced TNF receptor 1 (TNFR1) inhibition was investigated with the aid of protein synthesis, co-immunoprecipitation, and cytokine arrays. Notably, brusatol inhibited TNFR1 protein synthesis and suppressed both the canonical nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway and TNF-α-induced cytokine secretion. We further tested the functional effect of brusatol on atherosclerosis development in vivo using two different atherosclerosis mouse models, specifically, acute partial carotid ligation and conventional chronic high-fat diet-fed mouse models. Administration of brusatol led to significant suppression of atherosclerosis development in both mouse models. Our finding that brusatol prevents atherosclerosis via inhibition of TNFR1 protein synthesis supports the potential of downregulation of cell surface TNFR1 as an effective therapeutic approach to inhibit development of atherosclerosis., (© 2021 Federation of American Societies for Experimental Biology.)

To investigate whether the proteasome subunit alpha 6 (PSMA6) gene has an effect on fat deposition, the gene expression profile was first detected in Berkshire pigs and Jinhua pigs (JHP). The results demonstrated that significantly higher levels of mRNA expression were identified in adipose tissues and the liver. Interestingly, when compared to the longissimus dorsi muscle (LDM) in each breed, it was discovered that the expression levels of the PSMA6 gene in these tissues of JHP were considerably higher than those in Berkshire pigs. Meantime, some significant correlations of PSMA6 mRNA expression in lipid metabolism-related tissues such as the liver and LDM with the marbling score, as well as the content of intramuscular fat (IMF), in pigs were found by correlation coefficient analysis. To further explore the effects of PSMA6 expression on fat deposition, we performed PSMA6 overexpression in 3T3-L1 cells via Lentivirus infection. Our results indicated that PSMA6 could promote cell proliferation and accelerate cell division. It was also found that the transcription factors CCAAT/enhancer-binding protein alpha (CEBPA) and peroxisome proliferator-activated receptor gamma (PPARG), as well as the key genes related to adipogenesis, were upregulated, while the genes related to fat oxidation were significantly downregulated, which indicated that the PSMA6 gene could stimulate the differentiation of preadipocytes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The proteasome is a multi-subunit complex and a major proteolytic machinery in cells. Most subunits are essential for proteasome function, and depletion of individual subunits normally results in lethality. RPN-12/Rpn12/PSMD8 is a lid subunit of the 19S regulatory particle (RP) of the 26S proteasome. Studies in Caenorhabditis elegans demonstrated that RNAi depletion of RPN-12 does not result in lethality. RPN-12 has not been well studied in higher eukaryotes. In this study, we investigate the biological significance of RPN-12 in C. elegans., Results: We found that the null mutant rpn-12(av93) did not cause major impairment of the proteolytic activity of the proteasome. Most rpn-12(av93) hermaphrodites lack sperm leading to feminization of the germ line that can be partially rescued by mating to males. The lack of sperm phenotype can be suppressed by downregulation of TRA-1, a player in the hermaphrodite germline sex determination pathway. Also, rpn-12(av93) animals show significant nuclear accumulation of the meiotic kinase WEE-1.3, a protein predominantly localized to the perinuclear region. Interestingly, chemical inhibition of the proteasome did not cause nuclear accumulation of WEE-1.3., Conclusions: RPN-12 plays a previously unknown role in oogenesis and the germline sex determination pathway in C. elegans hermaphrodites., (© 2020 Wiley Periodicals LLC.)

Viruses confiscate cellular components of the ubiquitin-proteasome system (UPS) to facilitate many aspects of the infectious cycle. The 26S proteasome is an ATP-dependent, multisubunit proteolytic machine present in all eukaryotic cells. The proteasome executes the controlled degradation of functional proteins, as well as the hydrolysis of aberrantly folded polypeptides. There is growing evidence for the role of the UPS in viral entry. The UPS assists in several steps of the initiation of infection, including endosomal escape of the entering virion, intracellular transport of incoming nucleocapsids and uncoating of the viral genome. Inhibitors of proteasome activity, including MG132, epoxomicin, lactacystin and bortezomib have been integral to developments in this area. Here, we review the mechanistic details of UPS involvement in the entry process of viruses from a multitude of families. The possibility of proteasome inhibitors as therapeutic antiviral agents is highlighted., (© 2020 John Wiley & Sons Ltd.)

Alzheimer's disease (AD) is characterized by proteasome activity impairment, oxidative stress, and epigenetic changes, resulting in β -amyloid (A β ) production/degradation imbalance. Apolipoprotein E (ApoE) is implicated in A β clearance, and particularly, the ApoE ε 4 isoform predisposes to AD development. Regular physical activity is known to reduce AD progression. However, the impact of ApoE polymorphism and physical exercise on A β production and proteasome system activity has never been investigated in human peripheral blood cells, particularly in erythrocytes, an emerging peripheral model used to study biochemical alteration. Therefore, the influence of ApoE polymorphism on the antioxidant defences, amyloid accumulation, and proteasome activity was here evaluated in human peripheral blood cells depending on physical activity, to assess putative peripheral biomarkers for AD and candidate targets that could be modulated by lifestyle. Healthy subjects were enrolled and classified based on the ApoE polymorphism (by the restriction fragment length polymorphism technique) and physical activity level (Borg scale) and grouped into ApoE ε 4/non- ε 4 carriers and active/non-active subjects. The plasma antioxidant capability (AOC), the erythrocyte A β production/accumulation, and the nuclear factor erythroid 2-related factor 2 (Nrf2) mediated proteasome functionality were evaluated in all groups by the chromatographic and immunoenzymatic assay, respectively. Moreover, epigenetic mechanisms were investigated considering the expression of histone deacetylase 6, employing a competitive ELISA, and the modulation of two key miRNAs (miR-153-3p and miR-195-5p), through the miRNeasy Serum/Plasma Mini Kit. ApoE ε 4 subjects showed a reduction in plasma AOC and an increase in the Nrf2 blocker, miR-153-3p, contributing to an enhancement of the erythrocyte concentration of A β . Physical exercise increased plasma AOC and reduced the amount of A β and its precursor, involving a reduced miR-153-3p expression and a miR-195-5p enhancement. Our data highlight the impact of the ApoE genotype on the amyloidogenic pathway and the proteasome system, suggesting the positive impact of physical exercise, also through epigenetic mechanisms., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Rebecca Piccarducci et al.)

Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Proteinaceous substrates are tagged with polyUb and shuttle proteins, and these signals are then recognized by the proteasome, which subsequently degrades the substrate. To date, three proteasomal receptors have been identified, as well as multiple shuttle proteins and numerous types of polyUb chains that signal for degradation. While the components of this pathway are well-known, our understanding of their interplay is unclear-especially in the context of Rpn1, the largest proteasomal subunit. Here, using nuclear magnetic resonance (NMR) spectroscopy in combination with competition assays, we show that Rpn1 associates with UBL-containing proteins and polyUb chains, while exhibiting a preference for shuttle protein Rad23. Rpn1 appears to contain multiple Ub/UBL-binding sites, theoretically as many as one for each of its hallmark proteasome/cyclosome repeats. Remarkably, we also find that binding sites on Rpn1 can be shared among Ub and UBL species, while proteasomal receptors Rpn1 and Rpn10 can compete with each other for binding of shuttle protein Dsk2. Taken together, our results rule out the possibility of exclusive recognition sites on Rpn1 for individual Ub/UBL signals and further emphasize the complexity of the redundancy-laden proteasomal degradation pathway., Competing Interests: Conflict 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.)

The ubiquitin-proteasome system is the major pathway for the maintenance of protein homeostasis. Its inhibition causes accumulation of ubiquitinated proteins; this accumulation has been associated with several of the most common neurodegenerative diseases. Several genetic factors have been identified for most neurodegenerative diseases, however, most cases are considered idiopathic, thus making the study of the mechanisms of protein accumulation a relevant field of research. It is often mentioned that the biggest risk factor for neurodegenerative diseases is aging, and several groups have reported an age-related alteration of the expression of some of the 26S proteasome subunits and a reduction of its activity. Proteasome subunits interact with proteins that are known to accumulate in neurodegenerative diseases such as α-synuclein in Parkinson's, tau in Alzheimer's, and huntingtin in Huntington's diseases. These interactions have been explored for several years, but only until recently, we are beginning to understand them. In this review, we discuss the known interactions, the underlying patterns, and the phenotypes associated with the 26S proteasome subunits in the etiology and progression of neurodegenerative diseases where there is evidence of proteasome involvement. Special emphasis is made in reviewing proteasome subunits that interact with proteins known to have an age-related altered expression or to be involved in neurodegenerative diseases to explore key effectors that may trigger or augment their progression. Interestingly, while the causes of age-related reduction of some of the proteasome subunits are not known, there are specific relationships between the observed neurodegenerative disease and the affected proteasome subunits., (Copyright © 2020 IMSS. Published by Elsevier Inc. All rights reserved.)

Accumulating evidence indicates that the dysregulation of the miRNAs/mRNA-mediated carcinogenic signaling pathway network is intimately involved in glioma initiation and progression. In the present study, by performing experiments and bioinformatics analysis, we found that RPN2 was markedly elevated in glioma specimens compared with normal controls, and its upregulation was significantly linked to WHO grade and poor prognosis. Knockdown of RPN2 inhibited tumor proliferation and invasion, promoted apoptosis, and enhanced temozolomide (TMZ) sensitivity in vitro and in vivo. Mechanistic investigation revealed that RPN2 deletion repressed β-catenin/Tcf-4 transcription activity partly through functional activation of glycogen synthase kinase-3β (GSK-3β). Furthermore, we showed that RPN2 is a direct functional target of miR-181c. Ectopic miR-181c expression suppressed β-catenin/Tcf-4 activity, while restoration of RPN2 partly reversed this inhibitory effect mediated by miR-181c, implying a molecular mechanism in which TMZ sensitivity is mediated by miR-181c. Taken together, our data revealed a new miR-181c/RPN2/wnt/β-catenin signaling axis that plays significant roles in glioma tumorigenesis and TMZ resistance, and it represents a potential therapeutic target, especially in GBM.

Cutaneous T-cell lymphomas (CTCLs) represent a rare form of non-Hodgkin lymphomas characterized by an accumulation of malignant CD4 + T cells in the skin. TP53 genetic alteration is one of the most prevalent genetic abnormalities in CTCLs. Therefore, it is a promising target for innovative therapeutic approaches. We found that p53 could physically interact with histone deacetylase (HDAC) 1 and HDAC8, and was subsequently deacetylated to lose its function in CTCL cells, and the p53 downstream apoptosis-associated genes were repressed. Thus, the anti-CTCL activity displayed by HDAC inhibitors depends on p53 status. However, recent studies have reported that HDAC inhibitors could induce a wide variety of drug-resistant characteristics in cancer cells by regulating ATP-binding cassette transporters. Moreover, we discovered that Baicalein, a natural product, exhibited an inhibitory effect on HDAC1 and HDAC8. Though the inhibition of HDAC1 was mild, Baicalein could induce the degradation of HDAC1 through the ubiquitin proteasome pathway, thereby markedly upregulating the acetylation of histone H3 without promoting ATP-binding cassette transporter gene expression. In terms of the mechanism, Baicalein showed better growth inhibition than traditional HDAC inhibitors in CTCLs. This study indicates a special mechanism of HDAC1 and HDAC8 and p53 in T-cell lymphoma cells and identifies a potential and safe natural HDAC inhibitor for the treatment of CTCLs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Pathological cardiac hypertrophy eventually leads to heart failure without adequate treatment. REGγ is emerging as 11S proteasome activator of 20S proteasome to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner. Here, we found that REGγ was significantly upregulated in the transverse aortic constriction (TAC)-induced hypertrophic hearts and angiotensin II (Ang II)-treated cardiomyocytes. REGγ deficiency ameliorated pressure overload-induced cardiac hypertrophy were associated with inhibition of cardiac reactive oxygen species (ROS) accumulation and suppression of protein phosphatase 2A catalytic subunit α (PP2Acα) decay. Mechanistically, REGγ interacted with and targeted PP2Acα for degradation directly, thereby leading to increase of phosphorylation levels and nuclear export of Forkhead box protein O (FoxO) 3a and subsequent of SOD2 decline, ROS accumulation, and cardiac hypertrophy. Introducing exogenous PP2Acα or SOD2 to human cardiomyocytes significantly rescued the REGγ-mediated ROS accumulation of Ang II stimulation in vitro. Furthermore, treatment with superoxide dismutase mimetic, MnTBAP prevented cardiac ROS production and hypertrophy features that REGγ caused in vivo, thereby establishing a REGγ-PP2Acα-FoxO3a-SOD2 pathway in cardiac oxidative stress and hypertrophy, indicates modulating the REGγ-proteasome activity may be a potential therapeutic approach in cardiac hypertrophy-associated disorders.

Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno-associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well-known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood-derived CD34 + HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype-dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector-mediated transgene expression. Taken together, these studies expand our understanding of rAAV6-mediated transduction in hematopoietic cells and are informative for improving rAAV6-based treatment of blood diseases., (© 2020 Federation of American Societies for Experimental Biology.)

Background: Intracellular lipid accumulation is associated with various diseases, particularly cancer. Mitochondrial dysfunction is considered as a cause of lipid accumulation; however, the related underlying mechanism remains unclear., Findings: We found that Von Hippel-Lindau (VHL)-deficiency led to lipid accumulation and mitochondrial dysfunction in renal cell carcinoma cells. Moreover, VHL downregulated ATP-citrate lyase (ACLY), a key enzyme in de novo lipid synthesis, at the transcriptional level, which inhibited intracellular lipid accumulation in human renal carcinoma tissues. We identified PPARγ as the transcription factor regulating ACLY expression by binding to the cis-regulatory site PPRE on its promoter. VHL directly interacted with and promoted ubiquitination of PPARγ, leading to its degradation both in vitro and in vivo, resulting in the downregulation of ACLY. Furthermore, adenovirus-mediated VHL overexpression substantially ameliorated hepatic steatosis induced by a high-fat diet in db/db mice. Importantly, low VHL expression was associated with high ACLY expression and poor prognosis in human liver carcinoma in a dataset in The Cancer Genome Atlas., Conclusions: VHL plays role in cellular lipid metabolism via regulating mitochondria and targeting PPARγ, a transcription factor for ACLY independent of hypoxia-inducible factor 1α. A novel VHL-PPARγ-ACLY axis and its implication in fatty liver disease and cancer were uncovered., Competing Interests: Declaration of competing interest No potential conflicts of interest relevant to this article were reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sulfolobus acidocaldarius is the closest experimentally tractable archaeal relative of eukaryotes and, despite lacking obvious cyclin-dependent kinase and cyclin homologs, has an ordered eukaryote-like cell cycle with distinct phases of DNA replication and division. Here, in exploring the mechanism of cell division in S. acidocaldarius , we identify a role for the archaeal proteasome in regulating the transition from the end of one cell cycle to the beginning of the next. Further, we identify the archaeal ESCRT-III homolog, CdvB, as a key target of the proteasome and show that its degradation triggers division by allowing constriction of the CdvB1:CdvB2 ESCRT-III division ring. These findings offer a minimal mechanism for ESCRT-III-mediated membrane remodeling and point to a conserved role for the proteasome in eukaryotic and archaeal cell cycle control., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Aims: Liver cancer is one of the leading causes of cancer death worldwide owing to its delayed diagnosis and absence of efficient treatment at advanced TNM stages. Increasing evidence demonstrated that microRNAs are implicated in tumorgenesis and cancer development by regulating cancer-related proteins. This study aimed to explore the effect of miR-3619-5p on cell growth in liver cancer., Main Methods: The effect of miR-3619-5p on cell proliferation was measured by quantitative real-time PCR, MTT assay, flow cytometry, and Immunofluorescence assay. The interaction between miR-3619-5p and PSMD10 was validated using dual-luciferase. The expression of PSMD10 and Ki67 was further determined by immunohistochemistry., Key Findings: MiR-3619-5p over-expression remarkably inhibited cell proliferation and induced G1 phase arrest, accompanied with reduced expression of proliferating cell nuclear antigen. The expression of miR-3619-5p was negatively correlated to that of PSMD10, and PSMD10 was validated to be a downstream target of miR-3619-5p. Moreover, miR-3619-5p induced suppressed proliferation and G1 phase arrest were abrogated by elevated the expression of PSMD10 in liver cancer cells. PSMD10 over-expression also induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) and retinoblastoma protein (Rb1). Besides, elevated cyclin A, cyclin D1 and cyclin E expression supported that PSMD10 promoted the progress of cell cycle. In addition, miR-3619-5p inhibited tumor growth in vivo by targeting PSMD10, accompanied with blocked cell cycle., Significance: In conclusion, our findings revealed that miR-3619-5p inhibits cancer cell proliferation by targeting PSMD10, and miR-3619-5p as a potential therapeutic target for the treatment of liver cancer., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms., Methods: MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis., Results: Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis., Conclusions: Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII., (Copyright © 2020 by the American Society of Nephrology.)

Rationale: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and autophagic-lysosomal pathway defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the 2 catabolic pathways will help advance cardiac pathophysiology and medicine., Objective: Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac autophagic-lysosomal pathway., Methods and Results: Myocardial macroautophagy, TFEB (transcription factor EB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of Psmc1 (an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediated Molcn1 silencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from wild type mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with wild type mice., Conclusions: (1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1 (mucolipin1)-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac autophagic-lysosomal pathway activation during proteasome malfunction.

Brain aging is accompanied by an accumulation of damaged proteins, which results from deterioration of cellular quality control mechanisms and decreased protein degradation. The ubiquitin-proteasome system (UPS) is the primary proteolytic mechanism responsible for targeted degradation. Recent work has established a critical role of the UPS in memory and synaptic plasticity, but the role of the UPS in age-related cognitive decline remains poorly understood. Here, we measured markers of UPS function and related them to fear memory in rats. Our results show that age-related memory deficits are associated with reductions in phosphorylation of the Rpt6 proteasome regulatory subunit and corresponding increases in lysine-48 (K48)-linked ubiquitin tagging within the basolateral amygdala. Increases in K48 polyubiquitination were also observed in the medial prefrontal cortex and dorsal hippocampus. These data suggest that protein degradation is a critical component of age-related memory deficits. This extends our understanding of the relationship between the UPS, aging, and memory, which is an important step toward the prevention and treatment of deficits associated with normal cognitive aging and memory-related neurodegenerative diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Porcine circovirus 3 (PCV3) has been detected in major pig-producing countries around the world since its first report in the US in 2016. Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs. We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid (Cap) protein expression on autophagic response in human embryonic kidney cell line 293T (HEK293T). We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin (mTOR) in HEK293T cells. The ubiquitin-proteasome pathway is also involved in the autophagy process. These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Recognition of danger signals by a cell initiates a powerful cascade of events generally leading to inflammation. Inflammatory caspases and several other proteases become activated and subsequently cleave their target proinflammatory mediators. The irreversible nature of this process implies that the newly generated proinflammatory fragments need to be sequestered, inhibited, or degraded in order to cancel the proinflammatory program or prevent chronic inflammation. The Arg/N-degron pathway is a ubiquitin-dependent proteolytic pathway that specifically degrades protein fragments bearing N-degrons, or destabilizing residues, which are recognized by the E3 ligases of the pathway. Here, we report that the Arg/N-degron pathway selectively degrades a number of proinflammatory fragments, including some activated inflammatory caspases, contributing in tuning inflammatory processes. Partial ablation of the Arg/N-degron pathway greatly increases IL-1β secretion, indicating the importance of this ubiquitous pathway in the initiation and resolution of inflammation. Thus, we propose a model wherein the Arg/N-degron pathway participates in the control of inflammation in two ways: in the generation of inflammatory signals by the degradation of inhibitory anti-inflammatory domains and as an "off switch" for inflammatory responses through the selective degradation of proinflammatory fragments.

The ubiquitin-proteasome system is a master regulator of neural development and the maintenance of brain structure and function. It influences neurogenesis, synaptogenesis, and neurotransmission by determining the localisation, interaction, and turnover of scaffolding, presynaptic, and postsynaptic proteins. Moreover, ubiquitin-proteasome system signalling transduces epigenetic changes in neurons independently of protein degradation and, as such, dysfunction of components and substrates of this system has been linked to a broad range of brain conditions. Although links between ubiquitin-proteasome system dysfunction and neurodegenerative disorders have been known for some time, only recently have similar links emerged for neurodevelopmental disorders, such as schizophrenia. Here, we review the components of the ubiquitin-proteasome system that are reported to be dysregulated in schizophrenia, and discuss specific molecular changes to these components that might, in part, explain the complex causes of this mental disorder., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Acute myeloid leukemia is an aggressive myeloid malignancy, characterized by rapid cellular proliferation and generally high mortality. Due to the lack of a complete understanding of AML, its clinical outcomes are still not satisfactory. In this study, we examined the function of the long non-coding RNA-HLA complex P5 (HCP5) on AML by analyzing the clinical samples, TCGA data, and by shRNA-mediated HCP5 deficiency in vitro. Our results showed that HCP5 is highly expressed in AML and is positive associated with poor prognosis, and HCP5 knockdown was significantly suppressing AML cell line proliferation and inducing G1/S arrest in vitro. In mechanism, the proteasome subunit beta type 8 (PSMB8) expression was dramatically inhibited in HCP5 knockdown cells while increased in HCP5 overexpression cells. PSMB8 was also highly expressed in AML and with poor prognosis. Furthermore, HCP5 regulates PI3K/AKT pathway activation depending on PSMB8. Our results showed a promoting function of HCP5 on AML and may provide a compelling biomarker and therapy target for AML.

Proteasomes are the main producers of antigenic peptides presented to CD8 + T cells. They can cut proteins and release their fragments or recombine non-contiguous fragments thereby generating novel sequences, i.e. spliced peptides. Understanding which are the driving forces and the sequence preferences of both reactions can streamline target discovery in immunotherapies against cancer, infection and autoimmunity. Here, we present a large database of spliced and non-spliced peptides generated by proteasomes in vitro, which is available as simple CSV file and as a MySQL database. To generate the database, we performed in vitro digestions of 55 unique synthetic polypeptide substrates with different proteasome isoforms and experimental conditions. We measured the samples using three mass spectrometers, filtered and validated putative peptides, identified 22,333 peptide product sequences (15,028 spliced and 7,305 non-spliced product sequences). Our database and datasets have been deposited to the Mendeley (doi:10.17632/nr7cs764rc.1) and PRIDE (PXD016782) repositories. We anticipate that this unique database can be a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing, with various future translational applications.

In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.

Endemic arsenism is widely distributed in the world, which can damage multiple organs, especially in skin and liver. The etiology is clear, but the mechanisms involved remain unknown. Ubiquitin-proteasome pathway (UPP) is the main pathway regulating protein degradation of which proteasome subunit beta type-5(PSMB5) plays a dominant role. This paper aims to study the role and mechanism of PSMB5 in sodium arsenite (NaAsO 2 )-induced oxidative stress liver injury in L-02 cells. Firstly, L-02 cells were exposed to different concentrations of NaAsO 2 to establish a liver injury model of oxidative stress, and then mechanisms of oxidative stress were studied with carbobenzoxyl-leucyl-leucl-leucll-line (MG132) and knockdown PSMB5 (PSMB5-siRNA). The oxidative stress indicators, levels of 20S proteasome, the transcription and protein expression levels of PSMB5, Cu-Zn superoxide dismutase (SOD1), and glutathione peroxidase 1 (GPx1) were detected. The results demonstrated that NaAsO 2 could induce oxidative stress-induced liver injury and the activity of 20S proteasome and the protein expression of PSMB5, SOD1, and GPx1 decreased. After MG132 or PSMB5-siRNA pretreatment, the gene expression of PSMB decreased. After MG132 or PSMB5-siRNA pretreatment, and then L-02 cells were treated with NaAsO 2 , the gene expression of PSMB remarkably decreased; however, the protein expression of SOD1 and GPx1 increased. Overall, NaAsO 2 exposure could induce oxidative stress liver injury and low expression of PSMB5 in L-02 cells, and PSMB5 might play an important role in the regulation of oxidative stress by regulating the expression of SOD1 and Gpx1.

MyD88 is a conserved intracellular adaptor, which plays an important role in the innate immune system. MyD88 transmits signals for downstream of toll-like and IL-1 receptors to activate NF-κB signaling pathway, which is tightly controlled in the immune response to maintain immune intensity and immune homeostasis at different stages. NF-κB signaling pathway has been extensively studied in mammals, but regulatory molecular mechanism is still unclear in teleost fish. We determined that IRF3 and IRF8 can regulate MyD88-mediated NF-κB signaling pathway in fish. Interestingly, MyD88 is precisely regulated by IRF3 and IRF8 through the same mechanism but in completely opposite ways. IRF3 promotes MyD88-mediated NF-κB signaling pathway, whereas IRF8 inhibits the signaling pathway. MyD88 is regulated via ubiquitin-proteasome degradation, whereas IRF3 or IRF8 inhibited or promoted MyD88 degradation in this pathway. Specifically, in the early stage of lipopolysaccharide (LPS) stimulation or Vibrio infection, up-regulation of IRF3 and down-regulation of IRF8 eventually increased MyD88 expression to activate the NF-κB signaling pathway to trigger immune response. In the late stage of stimulation, down-regulated IRF3 and up-regulated IRF8 synergistically regulate the expression of MyD88 to a normal level, thus maintaining the immune balance of homeostasis and preventing serious damage from persistent over-immunization. This study presents information on Myd88-NF-κB signaling pathway in teleost fish and provides new insights into its regulatory mechanism in fish immune system., (Copyright © 2020 Yan, Zhao, Huo and Xu.)

The melanoma-associated antigen family A (MAGEA) antigens are expressed in a wide variety of malignant tumors but not in adult somatic cells, rendering them attractive targets for cancer immunotherapy. Here we show that a number of cancer-associated MAGEA mutants that undergo proteasome-dependent degradation in vitro could negatively impact their utility as immunotherapeutic targets. Importantly, in pancreatic ductal adenocarcinoma cell models, MAGEA6 suppresses macroautophagy (autophagy). The inhibition of autophagy is released upon MAGEA6 degradation, which can be induced by nutrient deficiency or by acquisition of cancer-associated mutations. Using xenograft mouse models, we demonstrated that inhibition of autophagy is critical for tumor initiation whereas reinstitution of autophagy as a consequence of MAGEA6 degradation contributes to tumor progression. These findings could inform cancer immunotherapeutic strategies for targeting MAGEA antigens and provide mechanistic insight into the divergent roles of MAGEA6 during pancreatic cancer initiation and progression., Competing Interests: YT, YW, KK, CG, OZ, TD, HL, NV, VB, DM, SE, HL, GM, KS No competing interests declared, (© 2020, Tsang et al.)

The majority of gastrointestinal stromal tumors (GISTs) are driven by oncogenic KIT signaling and can therefore be effectively treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate. However, most GISTs develop imatinib resistance through secondary KIT mutations. The type of resistance mutation determines sensitivity to approved second-/third-line TKIs but shows high inter- and intratumoral heterogeneity. Therefore, therapeutic strategies that target KIT independently of the mutational status are intriguing. Inhibiting the ubiquitin-proteasome machinery with bortezomib is effective in GIST cells through a dual mechanism of KIT transcriptional downregulation and upregulation of the pro-apoptotic histone H2AX but clinically problematic due to the drug's adverse effects. We therefore tested second-generation inhibitors of the 20S proteasome (delanzomib, carfilzomib and ixazomib) with better pharmacologic profiles as well as compounds targeting regulators of ubiquitination (b-AP15, MLN4924) for their effectiveness and mechanism of action in GIST. All three 20S proteasome inhibitors were highly effective in vitro and in vivo, including in imatinib-resistant models. In contrast, b-AP15 and MLN4924 were only effective at high concentrations or had mostly cytostatic effects, respectively. Our results confirm 20S proteasome inhibitors as promising strategy to overcome TKI resistance in GIST, while highlighting the complexity of the ubiquitin-proteasome machinery as a therapeutic target.

Key Message: Protein degradation is essential in plant growth and development. The stability of Cullin3 substrate adaptor protein BPM1 is regulated by multiple environmental cues pointing on manifold control of targeted protein degradation. A small family of six MATH-BTB genes (BPM1-6) is described in Arabidopsis thaliana. BPM proteins are part of the Cullin E3 ubiquitin ligase complexes and are known to bind at least three families of transcription factors: ERF/AP2 class I, homeobox-leucine zipper and R2R3 MYB. By targeting these transcription factors for ubiquitination and subsequent proteasomal degradation, BPMs play an important role in plant flowering, seed development and abiotic stress response. In this study, we generated BPM1-overexpressing plants that showed an early flowering phenotype, resistance to abscisic acid and tolerance to osmotic stress. We analyzed BPM1-GFP protein stability and found that the protein has a high turnover rate and is degraded by the proteasome 26S in a Cullin-dependent manner. Finally, we found that BPM1 protein stability is environmentally conditioned. Darkness and salt stress triggered BPM1 degradation, whereas elevated temperature enhanced BPM1 stability and accumulation in planta.

While chronic restraint stress (CRS) results in depression-like behaviors possibly through oxidative stress in the brain, its molecular etiology and the development of therapeutic strategies remain elusive. Since oxidized proteins can be targeted by the ubiquitin-proteasome system, we investigated whether increased proteasome activity might affect the stress response in mice. Transgenic mice, expressing the N-terminally deleted version of α3 subunit (α3ΔN) of the proteasome, which has been shown to generate open-gated mutant proteasomes, in the forebrain were viable and fertile, but showed higher proteasome activity. After being challenged with CRS for 14 d, the mutant mice with hyperactive proteasomes showed significantly less immobility time in the forced swimming test compared with their wild-type littermates, suggesting that the α3ΔN transgenic mice are resistant to CRS. The accumulation of ER stress markers, such as polyubiquitin conjugates and phospho-IRE1α, was also significantly delayed in the hippocampus of the mutants. Notably, α3ΔN mice exhibited little deficits in other behavioral tasks, suggesting that stress resilience is likely due to the degradation of misfolded proteins by the open-gated proteasomes. These data strongly indicate that not only is the proteasome a critical modulator of stress response in vivo but also a possible therapeutic target for reducing chronic stress.

Motor neuron diseases are neurodegenerative diseases that are characterized by degeneration of the upper and lower motor neurons in the central nervous system. Mutations in Dynactin 1 (DCTN1), a component in the Dynein/Dynactin motor complex, have been previously identified to cause motor neuron diseases and other neurodegenerative disorders. Recent studies showed that motor neuron disease-linked mutation, such as G59S mutation, could lead to dysfunction and protein aggregation of DCTN1. However, the cellular pathway involved in the clearance of DCTN1 aggregates is still not fully elucidated. In this study, we employed a culture cell model of DCTN1-linked neurodegeneration and explored the role of cellular protein control systems in the regulation of wild type and mutant DCTN1. We find that the ubiquitin-proteasome system, but not autophagy, is the primary protein degradation system for the turnover of both wild type and G59S DCTN1 under normal conditions. However, it turns out that autophagy can play a role in the clearance of protein aggregates of G59S DCTN1 when the proteasome activity is inhibited. Importantly, overexpression of TFEB, a master regulator of autophagy, promotes the autophagic clearance of G59S DCTN1 aggregates and ameliorates G59S DCTN1-induced cytotoxicity when the proteasomes are impaired. In conclusion, autophagy may play as a backup system to protect cells against the cytotoxicity induced by aggregate-prone DCTN1 when proteasomal function is damaged.

Background: As one of the most commonly diagnosed cancers among men and women, Colorectal Cancer (CRC) leads to high rates of morbidity and mortality across the globe. Recent anti- CRC therapies are now targeting specific signaling pathways involved in colorectal carcinogenesis. Ubiquitin Proteasome System (UPS) and autophagy are two main protein quality control systems, which play major roles in the carcinogenesis of colorectal cancer. A balanced function of these two pathways is necessary for the regulation of cell proliferation and cell death., Objective: In this systematic review, we discuss the available evidence regarding the roles of autophagy and ubiquitination in progression and inhibition of CRC., Methods: The search terms "colorectal cancer" or "colon cancer" or "colorectal carcinoma" or "colon carcinoma" in combination with "ubiquitin proteasome" and "autophagy" were searched in PubMed, Web of Science, and Scopus databases, and also Google Patents (https://patents.google .com) from January 2000 to Feb 2020., Results: The most important factors involved in UPS and autophagy have been investigated. There are many important factors involved in UPS and autophagy but this systematic review shows the studies that have mostly focused on the role of ATG, 20s proteasome and mTOR in CRC, and the more important factors such as ATG8, FIP200, and TIGAR factors that are effective in the regulation of autophagy in CRC cells have not been yet investigated., Conclusion: The most important factors involved in UPS and autophagy such as ATG, 20s proteasome and mTOR, ATG8, FIP200, and TIGAR can be considered in drug therapy for controlling or activating autophagy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

To achieve homeostasis, cells evolved dynamic and self-regulating quality control processes to adapt to new environmental conditions and to prevent prolonged damage. We discuss the importance of two major quality control systems responsible for degradation of proteins and organelles in eukaryotic cells: the ubiquitin-proteasome system (UPS) and autophagy. The UPS and autophagy form an interconnected quality control network where decision-making is self-organized on the basis of biophysical parameters (binding affinities, local concentrations, and avidity) and compartmentalization (through membranes, liquid-liquid phase separation, or the formation of aggregates). We highlight cellular quality control factors that delineate their differential deployment toward macromolecular complexes, liquid-liquid phase-separated subcellular structures, or membrane-bound organelles. Finally, we emphasize the need for continuous promotion of quantitative and mechanistic research into the roles of the UPS and autophagy in human pathophysiology., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)