Proteasome Activity Research Articles

Abstract Tu052: RPN6-Serine14 Phosphorylation by PKA Protects Against Systolic Overload-Induced Cardiac Remodeling and Heart Failure

The ubiquitin-proteasome system is the main proteolytic pathway for quality control (PQC) and regulatory degradation of proteins in living cells. Systolic overload, as seen in hypertension and aortic stenosis, is a leading cause of heart failure (HF). Proteasome functional insufficiency is implicated in HF caused by systolic overload. Previous studies have established that Ser14-RPN6 phosphorylation (pS14-RPN6) mediates the activation of 26S proteasomes by protein kinase A. However, the pathophysiological significance of pS14-RPN6 in systolic overload remains to be established. We hypothesize that pS14-RPN6 helps maintain proteostasis and protects the heart during systolic overload. We observed a significant increase in myocardial pS14-RPN6 and ubiquitin conjugates in human HF of ischemic or non-ischemic etiologies (p=0.020, p=0.034) and similarly in wild type (WT) mice after 2 weeks of transverse aortic constriction (TAC) (p<0.0001, p=0.001). To investigate the role of pS14-RPN6 in vivo, our lab created Rpn6 S14A (S14A) knock-in mice where Ser14-Rpn6 phosphorylation is blocked. Here we have compared the responses to TAC between WT and S14A mice. We found that TAC-induced increases in myocardial proteasome peptidase activities were significantly attenuated, and accumulation of ubiquitin conjugates exacerbated in S14A mice (p<0.0001; p<0.0005). Serial echocardiography revealed that S14A mice displayed significantly greater left ventricular (LV) end-diastolic posterior wall thickness and LV mass to body weight ratios at 1- and 12-week post-TAC (p=0.024, 0.0003; p=0.005, 0.01), and lower ejection fraction at 12-week post-TAC than WT mice (p=0.035). S14A mice also showed greater heart weight to tibial length (TL) and ventricular weight to TL ratios at 1 and 12-week post-TAC (1-week, p=0.043, 0.039; 12-week, p=0.007, 0.006). Wheat germ agglutinin and picrosirius red staining indicated a greater cardiomyocyte cross-sectional area and cardiac fibrosis in 12-week post-TAC S14A mice vs. WT mice (p<0.0001; p=0.005). These novel findings demonstrate compellingly that myocardial pS14-RPN6 helps maintain proteostasis, curtails cardiac hypertrophy and delays heart failure occurrence during systolic overload

Targeting the immunoproteasome in hypothalamic neurons as a novel therapeutic strategy for high-fat diet-induced obesity and metabolic dysregulation

ObjectiveObesity represents a significant global health challenge characterized by chronic low-grade inflammation and metabolic dysregulation. The hypothalamus, a key regulator of energy homeostasis, is particularly susceptible to obesity’s deleterious effects. This study investigated the role of the immunoproteasome, a specialized proteasomal complex implicated in inflammation and cellular homeostasis, during metabolic diseases.MethodsThe levels of the immunoproteasome β5i subunit were analyzed by immunostaining, western blotting, and proteasome activity assay in mice fed with either a high-fat diet (HFD) or a regular diet (CHOW). We also characterized the impact of autophagy inhibition on the levels of the immunoproteasome β5i subunit and the activation of the AKT pathway. Finally, through confocal microscopy, we analyzed the contribution of β5i subunit inhibition on mitochondrial function by flow cytometry and mitophagy assay.ResultsUsing an HFD-fed obese mouse model, we found increased immunoproteasome levels in hypothalamic POMC neurons. Furthermore, we observed that palmitic acid (PA), a major component of saturated fats found in HFD, increased the levels of the β5i subunit of the immunoproteasome in hypothalamic neuronal cells. Notably, the increase in immunoproteasome expression was associated with decreased autophagy, a critical cellular process in maintaining homeostasis and suppressing inflammation. Functionally, PA disrupted the insulin-glucose axis, leading to reduced AKT phosphorylation and increased intracellular glucose levels in response to insulin due to the upregulation of the immunoproteasome. Mechanistically, we identified that the protein PTEN, a key regulator of insulin signaling, was reduced in an immunoproteasome-dependent manner. To further investigate the potential therapeutic implications of these findings, we used ONX-0914, a specific immunoproteasome inhibitor. We demonstrated that this inhibitor prevents PA-induced insulin-glucose axis imbalance. Given the interplay between mitochondrial dysfunction and metabolic disturbances, we explored the impact of ONX-0914 on mitochondrial function. Notably, ONX-0914 preserved mitochondrial membrane potential and attenuated mitochondrial ROS production in the presence of PA. Moreover, we found that ONX-0914 reduced mitophagy in the presence of PA.ConclusionsOur findings strongly support the pathogenic involvement of the immunoproteasome in hypothalamic neurons in the context of HFD-induced obesity and metabolic disturbances. Targeting the immunoproteasome highlights a promising therapeutic strategy to mitigate the detrimental effects of obesity on the insulin-glucose axis and cellular homeostasis. This study provides valuable insights into the mechanisms driving obesity-related metabolic diseases and offers potential avenues for developing novel therapeutic interventions.

Abstract We126: Myocarditis Following mRNA Vaccination for COVID-19 is Mediated by CXCL10 and IFN-γ

Introduction: mRNA vaccines have shown remarkable efficacy against symptomatic COVID-19, reshaping the pandemic's trajectory. Despite a robust safety profile, ongoing monitoring is crucial due to the rare but concerning myocarditis cases, predominantly affecting males under 30. Symptoms appear around 2.6 days post-vaccination, with chest pain, shortness of breath, and palpitations. Elevated cTnI and ST-segment changes are common, along with late gadolinium enhancement in cardiac MRI. Understanding these rare adverse effects is essential for the expanding scope of mRNA technology, spanning infectious disease vaccines and anti-cancer immunotherapies. Hypothesis: Myocarditis linked to COVID-19 mRNA vaccines involves cytokine production from myeloid cells, leading to impaired cardiac function. Methodology: a, Reanalysis of proteomics data of healthy and myocarditis individuals following COVID-19 vaccination; b, Macrophages (N = 7 lines) were exposed to BNT162b2 or mRNA-1273, and analyzed by cytokine array and RNA-seq; c, Cardiomyocytes (N = 4 lines) were treated with CXCL10 (1 and 10 ng/mL) and IFN-γ (0.5 and 5 ng/mL), with or without genistein (10 μM), followed by functional assessment, transcriptomic and proteomic profiling; d, Mice (6 per group) were administered with CXCL10 and IFN-γ, with or without genistein pretreatment (7 days). Myeloid cell infiltration in the heart, serum cTnI levels, RNA-seq, and immunoproteasome activity were analyzed. Results: Examination of post-vaccination plasma revealed a notable rise in CXCL10 and IFN-γ levels in patients. Similarly, macrophages exposed to mRNA vaccines displayed heightened production of these cytokines. When cardiomyocytes were exposed to these cytokines, they exhibited compromised contractility, increased arrhythmogenicity, and gene expression patterns reminiscent of myocarditis. Genistein, an anti-inflammatory phytoestrogen, demonstrated significant alleviation of these effects, preventing cytokine-induced proteasomal activation and preserving contractile function. Mouse model of cytokine-induced myocarditis showed that genistein substantially reduced cardiac injury markers and immune cell infiltration. Conclusion: These findings highlight CXCL10 and IFN-γ as pivotal contributors to myocarditis following mRNA vaccination and suggest genistein as a potential therapeutic intervention to mitigate associated cardiovascular risks.

Floxuridine supports UPS independent of germline signaling and proteostasis regulators via involvement of detoxification in C. elegans.

The ubiquitin-proteasome system (UPS) is critical for maintaining proteostasis, influencing stress resilience, lifespan, and thermal adaptability in organisms. In Caenorhabditis elegans, specific proteasome subunits and activators, such as RPN-6, PBS-6, and PSME-3, are associated with heat resistance, survival at cold (4°C), and enhanced longevity at moderate temperatures (15°C). Previously linked to improving proteostasis, we investigated the impact of sterility-inducing floxuridine (FUdR) on UPS functionality under proteasome dysfunction and its potential to improve cold survival. Our findings reveal that FUdR significantly enhances UPS activity and resilience during proteasome inhibition or subunit deficiency, supporting worms' normal lifespan and adaptation to cold. Importantly, FUdR effect on UPS activity occurs independently of major proteostasis regulators and does not rely on the germ cells proliferation or spermatogenesis. Instead, FUdR activates a distinct detoxification pathway that supports UPS function, with GST-24 appearing to be one of the factors contributing to the enhanced activity of the UPS upon knockdown of the SKN-1-mediated proteasome surveillance pathway. Our study highlights FUdR unique role in the UPS modulation and its crucial contribution to enhancing survival under low-temperature stress, providing new insights into its mechanisms of action and potential therapeutic applications.

Selective recognition of PTRE1 transcripts mediated by protein–protein interaction between the m6A reader ECT2 and PTRE1

N6-methyladenosine (m6A) is a prevalent internal post-transcriptional modification in eukaryotic RNAs executed by m6A-binding proteins known as “readers.” Our previous research demonstrated that the Arabidopsis m6A reader ECT2 positively regulates transcript levels of the proteasome regulator PTRE1 and several 20S proteasome subunits, thereby enhancing 26S proteasome activity. However, mechanism underlying the selective recognition of m6A targets by readers, such as ECT2, remains elusive. In this study, we further demonstrate that ECT2 physically interacts with PTRE1 and several 20S proteasome subunits. This interaction, which occurs on the ribosome, involves the N terminus of PTRE1, suggesting that ECT2 might bind to the nascent PTRE1 polypeptide. Deleting ECT2’s protein interaction domain impairs its mRNA-binding ability, whereas mutations in the m6A-RNA-binding site do not affect protein–protein interactions. Moreover, introducing a novel protein-binding domain into ECT2 increases transcript levels of proteins interacting with this domain. Our findings indicate that interaction with the PTRE1 protein enhances ECT2’s binding to PTRE1 m6A mRNAs during translation, thereby regulating PTRE1 mRNA levels.

Identification of potent and reversible piperidine carboxamides that are species-selective orally active proteasome inhibitors to treat malaria

Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome β5 active-site (Pfβ5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto β7. Inhibition of Pfβ5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the β5/β6/β3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.

Proteasome activity inhibition mediates endoplasmic reticulum stress-apoptosis in triptolide/lipopolysaccharide-induced hepatotoxicity

Triptolide (TP) is a major active and toxic composition of the Chinese medicine Tripterygium wilfordii Hook. F. (TWHF), exhibiting various therapeutic bioactivities. Among the toxic effects, the hepatotoxicity of TP deserves serious attention. Previously, our research group proposed a new view of TP-related hepatotoxicity: hepatic hypersensitivity under lipopolysaccharide (LPS) stimulation. However, the mechanism of TP/LPS-induced hepatic hypersensitivity remains unclear. In this study, we investigated the mechanism underlying TP/LPS-induced hypersensitivity from the perspective of the inhibition of proteasome activity, activated endoplasmic reticulum stress (ERS)-related apoptosis, and the accumulation of reactive oxygen species (ROS). Our results showed that N-acetylcysteine (NAC), a common ROS inhibitor, decreased the expression of cleaved caspase-3 and cleaved PARP, which are associated with FLIP enhancement. Moreover, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was able to alleviate TP/LPS-induced hepatotoxicity by reducing ERS-related apoptosis protein expression (GRP78, p-eIF2α/eIF2α, ATF4, CHOP, cleaved caspase-3 and cleaved PARP) and ROS levels, with ATF4 being an indispensable mediator. In addition, the proteasome activity inhibitor MG-132 further aggravated ERS-related apoptosis, which indicated that the inhibition of proteasome activity also plays an important role in TP/LPS-related liver injuries. In summary, we propose that TP/LPS may upregulate the activation of ERS-associated apoptosis by inhibiting proteasome activity and enhancing ROS production through ATF4.Graphical

Establishment of a stable THP-1 cell line expressing PSMB9-eGFP-His protein and detection of immunoproteasome activity

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9-eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9-eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

Palm kernel meal regulates the expression of genes involved in the amino acid metabolism in the liver of Tibetan sheep

BackgroundPalm kernel meal (PKM) is a by-product of oil palm kernel after oil extraction, which is widely used in animal feeds due to its high energy content. This study aimed to investigate the impact of supplementing Tibetan sheep with PKM on their hepatic phenotype, oxidative stress and immune response. A total of 120 Tibetan lambs (Initial weight = 12.37 ± 0.92 kg) were randomly assigned into four groups: control group (C group, 0% PKM diet), low group (L group, 15% PKM diet), middle group (M group, 18% PKM diet), and high group (H group, 21% PKM diet) on a dry matter basis. The feeding experiment was performed for 130 d, including a 10 d adaption period.ResultsResults showed that the level of GSH-Px were higher in the H and M groups than in the C and L groups (P < 0.05). The levels of IgM and TNF-α were higher in the M group when compared to those on the C group (P < 0.05). The level of IgA was significantly higher in the M group than in the H group (P < 0.05). Additionally, compared with the others groups, the hepatocytes in the M group displayed a radial arrangement, forming hepatic plates that were centered around the central vein. The transcriptome results revealed that proteasome 26 S subunit, ATPase 3 (PSMC3), proteasome 26 S subunit, ATPase 5 (PSMC5), proteasome 26 S subunit ubiquitin receptor, non-ATPase 4 (PSMD4), proteasome activator subunit 1 (PSME1), acyl-CoA dehydrogenase short/branched chain (ACADSB), enoyl-CoA hydratase, short chain 1 (ECHS1), serine dehydratase (SDS), ornithine transcarbamylase (OTC), and phenylalanine hydroxylase (PAH) were the hub genes regulating the amino acid metabolism in the liver.ConclusionsIn summary, dietary 18% PMK supplementation contributed to improve the hepatic phenotype, oxidative stress and immune response through regulating the expression of related genes.

Primed for Interactions: Investigating the Primed Substrate Channel of the Proteasome for Improved Molecular Engagement.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

Targeting Microglial Immunoproteasome: A Novel Approach in Neuroinflammatory-Related Disorders.

It is widely acknowledged that the aging process is linked to the accumulation of damaged and misfolded proteins. This phenomenon is accompanied by a decrease in proteasome (c20S) activity, concomitant with an increase in immunoproteasome (i20S) activity. These changes can be attributed, in part, to the chronic neuroinflammation that occurs in brain tissues. Neuroinflammation is a complex process characterized by the activation of immune cells in the central nervous system (CNS) in response to injury, infection, and other pathological stimuli. In certain cases, this immune response becomes chronic, contributing to the pathogenesis of various neurological disorders, including chronic pain, Alzheimer's disease, Parkinson's disease, brain traumatic injury, and others. Microglia, the resident immune cells in the brain, play a crucial role in the neuroinflammatory response. Recent research has highlighted the involvement of i20S in promoting neuroinflammation, increased activity of which may lead to the presentation of self-antigens, triggering an autoimmune response against the CNS, exacerbating inflammation, and contributing to neurodegeneration. Furthermore, since i20S plays a role in breaking down accumulated proteins during inflammation within the cell body, any disruption in its activity could lead to a prolonged state of inflammation and subsequent cell death. Given the pivotal role of i20S in neuroinflammation, targeting this proteasome subtype has emerged as a potential therapeutic approach for managing neuroinflammatory diseases. This review delves into the mechanisms of neuroinflammation and microglia activation, exploring the potential of i20S inhibitors as a promising therapeutic strategy for managing neuroinflammatory disorders.

Dysregulated proteasome activity and steroid hormone biosynthesis are associated with mortality among patients with acute COVID-19

The persistence of coronavirus disease 2019 (COVID-19)-related hospitalization severely threatens medical systems worldwide and has increased the need for reliable detection of acute status and prediction of mortality. We applied a systems biology approach to discover acute-stage biomarkers that could predict mortality. A total 247 plasma samples were collected from 103 COVID-19 (52 surviving COVID-19 patients and 51 COVID-19 patients with mortality), 51 patients with other infectious diseases (IDCs) and 41 healthy controls (HCs). Paired plasma samples were obtained from survival COVID-19 patients within 1 day after hospital admission and 1–3 days before discharge. There were clear differences between COVID-19 patients and controls, as well as substantial differences between the acute and recovery phases of COVID-19. Samples from patients in the acute phase showed suppressed immunity and decreased steroid hormone biosynthesis, as well as elevated inflammation and proteasome activation. These findings were validated by enzyme-linked immunosorbent assays and metabolomic analyses in a larger cohort. Moreover, excessive proteasome activity was a prominent signature in the acute phase among patients with mortality, indicating that it may be a key cause of poor prognosis. Based on these features, we constructed a machine learning panel, including four proteins [C-reactive protein (CRP), proteasome subunit alpha type (PSMA)1, PSMA7, and proteasome subunit beta type (PSMB)1)] and one metabolite (urocortisone), to predict mortality among COVID-19 patients (area under the receiver operating characteristic curve: 0.976) on the first day of hospitalization. Our systematic analysis provides a novel method for the early prediction of mortality in hospitalized COVID-19 patients.

Promising Potential of Curcumin and Related Compounds for Antiviral Drug Discovery.

Viruses are acellular, microscopic, and mobile particles containing genetic particles, either DNA/RNA strands as nucleoproteins, responsible for 69,53,743 deaths till the year 2023. Curcumin and related compounds are among the areas of pivotal interest for researchers because of their versatile pharmacological profile. Chemically known as diferuloylmethane, which is a main constituent of turmeric along with demethoxycurcumin and bisdemethoxycurcumin, they have a broad spectrum of antiviral activity against viruses such as human immunodeficiency virus, herpes simplex virus, influenza virus (Avian influenza) and Hepatitis C virus HIV. The possible role of curcumin as an antiviral agent may be attributed to the activation of the 20S proteasome, a cellular machinery responsible for degrading unfolded or misfolded proteins in a ubiquitin-independent manner. It shows suppression of HBV entry at various infection stages by inhibiting cccDNA replication by inhibiting the Wnt/β-catenin signaling pathway to attenuate IAV-induced myocarditis.

PSME3 promotes lung adenocarcinoma development by regulating the TGF-β/SMAD signaling pathway.

Lung adenocarcinoma (LUAD) is one of the most common types of cancer worldwide. Proteasome activator subunit 3 (PSME3) is a subunit of a proteasome activator, and changes in PSME3 can lead to the development of many diseases in organisms. However, the specific mechanism of PSME3 in LUAD has not yet been elucidated. This study initially revealed the mechanism of PSME3 promoting the progression of lung adenocarcinoma, which provided a potential molecular target for clinical treatment. PSME3 expression in LUAD cells and tissues was assessed by bioinformatics analysis, immunohistochemistry (IHC), Western blotting (WB), and quantitative real time polymerase chain reaction (qRT-PCR). A series of functional experiments were used to evaluate the effects of PSME3 knockdown and overexpression on LUAD cell proliferation, migration, and apoptosis. The potential mechanism of PSME3 was explored by transcriptome sequencing and WB experiments. In this study, our initial findings indicated that PSME3 expression was abnormally high in LUAD and was associated with poor patient prognosis. Further, we found that the downregulation of PSME3 significantly inhibited LUAD cell proliferation, an effect that was verified by subcutaneous tumor formation experiments in nude mice. Similarly, the rate of invasion and migration of LUAD cells significantly decreased after the downregulation of PSME3. Using flow cytometry, we found that the knockdown of PSME3 caused cell cycle arrest at the G1/S phase. Through transcriptome sequencing, we found that the transforming growth factor-beta (TGF-β)/SMAD signaling pathway was closely related to LUAD, and we then validated the pathway using WB assays. We demonstrated that PSME3 was abnormally highly expressed in LUAD and related to poor patient prognosis; therefore, targeting PSME3 in the treatment of LUAD may represent a novel therapeutic approach.

Geraniol (GER) attenuated chronic sleep restriction (CSR)-induced neuroinflammation in adolescent mice

Sleep insufficiency is a significant health problem worldwide, and adolescent sleep restriction (SR) could induce multiple neurodevelopmental disorders in the central nervous system (CNS). Microglial-mediated neuroinflammation plays a vital role in multiple neurological diseases, and recent research showed the regulation effect of immunoproteasome on microglia functions. Geraniol (GER), an important ingredient in many essential oils, possesses diverse pharmacological properties like anti-inflammatory and antioxidant. The present study was designed to evaluate the neuroprotective effect of GER on SR in adolescent mice and further investigate the underlying mechanisms. Our results displayed that 14 days of chronic sleep restriction (CSR) induced cognitive decline, and anxiety-like and attention-deficit behaviors, which were mitigated by GER pretreatment. GER administration also reversed microglial pro-inflammatory response under CSR stimulation in the anterior cingulate cortex (ACC) regions by reducing the expression and secretion of cytokines like IL-1β and TNF-α. Mechanism research showed that LMP7 mRNA was selectively up-regulated under CSR treatment but down-regulated by GER administration. Proteasome activity and protein expression of LMP7 were consistent with mRNA data. ONX-0914 was applied to inhibit LMP7 selectively, and data validated that GER might alleviate CSR-induced neuroinflammation by regulating LMP7. Our study provides evidence that LMP7 is a critical regulator of CSR-induced proinflammation, and geraniol might be a promising therapy against CSR-induced neurodevelopmental disorders.

Transcriptomic Characterization of Key Factors and Signaling Pathways for the Regeneration of Partially Hepatectomized Liver in Zebrafish.

Liver regeneration induced by partial hepatectomy (PHx) has attracted intensive research interests due to the great significance for liver resection and transplantation. The zebrafish (Danio rerio) is an excellent model to study liver regeneration. In the fish subjected to PHx (the tip of the ventral lobe was resected), the lost liver mass could be fully regenerated in seven days. However, the regulatory mechanisms underlying the liver regeneration remain largely unknown. In this study, gene expression profiles during the regeneration of PHx-treated liver were explored by RNA sequencing (RNA-seq). The genes responsive to the injury of PHx treatment were identified and classified into different clusters based on the expression profiles. Representative gene ontology (GO) enrichments for the early responsive genes included hormone activity, ribosome biogenesis and rRNA processing, etc., while the late responsive genes were enriched in biological processes such as glutathione metabolic process, antioxidant activity and cellular detoxification. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments were also identified for the differentially expressed genes (DEGs) between the time-series samples and the sham controls. The proteasome was overrepresented by the up-regulated genes at all of the sampling time points. Inhibiting proteasome activity by the application of MG132 to the fish enhanced the expression of Pcna (proliferating cell nuclear antigen), an indicator of hepatocyte proliferation after PHx. Our data provide novel insights into the molecular mechanisms underlying the regeneration of PHx-treated liver.

PSMD11 promotes the proliferation of hepatocellular carcinoma by regulating the ubiquitination degradation of CDK4

BackgroundThe 26S proteasome non-ATPase regulatory subunit 11 is a multiprotein complex involved in the ATP-dependent degradation of ubiquitinated proteins, and PSMD11 plays a key role in the regulation of embryonic stem cell proteasome activity. However, the role of PSMD11 in hepatocellular carcinoma has not been studied. In this study, it was found that the expression of PSMD11 in HCC tissues was significantly higher than that in para-cancerous tissues, and was associated with poor prognosis. The results of in vitro experiments showed that PSMD11 knockdown could effectively inhibit the proliferation and apoptosis of hepatoma cell lines, and flow cytometry showed that the G0/G1 phase was significantly prolonged. Through protein spectrometry, immunoprecipitation and in vitro experiments, it was found that PSMD11 can promote the proliferation of hepatocellular carcinoma through regulating the ubiquitination of CDK4 and enhancing its protein stability. This study explores the mechanism of action of PSMD11 in hepatocellular carcinoma and provides new insights for the treatment of hepatocellular carcinoma.

SUMO protease and proteasome recruitment at the nuclear periphery differently affect replication dynamics at arrested forks.

Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components.

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate.

Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code.

The maintenance of oocytes in the mammalian ovary involves extreme protein longevity

Women are born with all of their oocytes. The oocyte proteome must be maintained with minimal damage throughout the woman’s reproductive life, and hence for decades. Here we report that oocyte and ovarian proteostasis involves extreme protein longevity. Mouse ovaries had more extremely long-lived proteins than other tissues, including brain. These long-lived proteins had diverse functions, including in mitochondria, the cytoskeleton, chromatin and proteostasis. The stable proteins resided not only in oocytes but also in long-lived ovarian somatic cells. Our data suggest that mammals increase protein longevity and enhance proteostasis by chaperones and cellular antioxidants to maintain the female germline for long periods. Indeed, protein aggregation in oocytes did not increase with age and proteasome activity did not decay. However, increasing protein longevity cannot fully block female germline senescence. Large-scale proteome profiling of ~8,890 proteins revealed a decline in many long-lived proteins of the proteostasis network in the aging ovary, accompanied by massive proteome remodeling, which eventually leads to female fertility decline.