Regulation Of Ubiquitin-proteasome System Research Articles

Bottom-up approach to deciphering the targets of the ubiquitin-proteasome system in porcine sperm capacitation

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

Unraveling Microbial Strategies: Targeting the Plant Ubiquitin - Proteasome Pathway

The Ubiquitin-Proteasome System (UPS) stands as a central regulator in the intricate web of plant immune responses, orchestrating the turnover of key immune components. Through the UPS, plants finely tune their defenses, from the initial recognition of pathogens to the activation of defense strategies. Immune receptors, signaling components associated with defense hormones, and transcription factors are among the targets modulated by the UPS, shaping both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). However, pathogens have evolved sophisticated strategies to manipulate the UPS for their benefit. Fungi, bacteria, and viruses deploy effector proteins to exploit the UPS, either by promoting the degradation of host defense elements or by interfering with UPS-mediated processes. This intricate interplay underscores the pivotal role of the UPS in plant-pathogen dynamics, presenting both challenges and opportunities for understanding and manipulating plant immunity. Unraveling the complexities of UPS regulation and pathogen exploitation thereof is crucial for advancing our knowledge of plant-microbe interactions and developing strategies for sustainable disease management in agriculture.

SARS-CoV-2 and UPS with potentials for therapeutic interventions

The Ubiquitin proteasome system (UPS), an essential eukaryotic/host/cellular post-translational modification (PTM), plays a critical role in the regulation of diverse cellular functions including regulation of protein stability, immune signaling, antiviral activity, as well as virus replication. Although UPS regulation of viral proteins may be utilized by the host as a defense mechanism to invade viruses, viruses may have adapted to take advantage of the host UPS. This system can be manipulated by viruses such as the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to stimulate various steps of the viral replication cycle and facilitate pathogenesis, thereby causing the respiratory disease COVID-19. Many SARS-CoV-2 encoded proteins including open reading frame 3a (ORF3a), ORF6, ORF7a, ORF9b, and ORF10 interact with the host's UPS machinery, influencing host immune signaling and apoptosis. Moreover, SARS-CoV-2 encoded papain-like protease (PLpro) interferes with the host UPS to facilitate viral replication and to evade the host’s immune system. These alterations in SARS-CoV-2 infected cells have been revealed by various proteomic studies, suggesting potential targets for clinical treatment. To provide insight into the underlying causes of COVID-19 and suggest possible directions for therapeutic interventions, this paper reviews the intricate relationship between SARS-CoV-2 and UPS. Promising treatment strategies are also investigated in this paper including targeting PLpro with zinc-ejector drugs, as well as targeting viral non-structural protein (nsp12) via heat treatment associated ubiquitin-mediated proteasomal degradation to reduce viral pathogenesis.

Ubiquitin-proteasome system regulation of a key gene regulatory factor, Paf1C

Paf1 (Polymerase-associated factor 1) complex (Paf1C) is evolutionarily conserved from yeast to humans, and facilitates transcription elongation as well as co-transcriptional histone covalent modifications and mRNA 3’-end processing. Thus, Paf1C is a key player in regulation of eukaryotic gene expression. Paf1C consists of Paf1, Cdc73, Ctr9, Leo1 and Rtf1 in both yeast and humans, but it has an additional component, Ski8, in humans. The abundances of these components regulate the assembly of Paf1C and/or its functions, thus implying the mechanisms involved in regulating the abundances of the Paf1C components in altered gene expression and hence cellular pathologies. Towards finding the mechanisms associated with the abundances of the Paf1C components, we analyzed here whether the Paf1C components are regulated via targeted ubiquitylation and 26S proteasomal degradation. We find that the Paf1C components except Paf1 do not undergo the 26S proteasomal degradation in both yeast and humans. Paf1 is found to be regulated by the ubiquitin-proteasome system (UPS) in yeast and humans. Alteration of such regulation changes Paf1’s abundance, leading to aberrant gene expression. Intriguingly, while the Rtf1 component of Paf1C does not undergo the 26S proteasomal degradation, it is found to be ubiquitylated, suggesting that Rtf1 ubiquitylation could be engaged in Paf1C assembly and/or functions. Collectively, our results reveal distinct UPS regulation of the Paf1C components, Paf1 and Rtf1, in a proteolysis-dependent and -independent manners, respectively, with functional implications.

2-Deoxy-d-ribose induces ferroptosis in renal tubular epithelial cells via ubiquitin-proteasome system-mediated xCT protein degradation

Ferroptosis is a novel form of cell death triggered by iron-dependent lipid peroxidation. Recent findings suggest that inhibiting system χc-induces ferroptosis by reducing intracellular cystine levels, and that ferroptosis in renal tubular epithelial cells (RTECs) contributes to acute kidney injury (AKI) and diabetic nephropathy. Moreover, 2-deoxy-d-ribose (dRib) has been shown to inhibit cystine uptake through xCT, the functional unit of system χc-, in β-cells. This study aimed to investigate if dRib induces ferroptosis in RTECs and identify the underlying mechanisms.dRib treatment reduced cystine uptake and glutathione (GSH) content, and increased intracellular levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), lipid reactive oxygen species (ROS), and cell death in both NRK-52E cells and primary cultured RTECs. However, treatment with inhibitors of ferroptosis, such as deferoxamine (DFO), ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1), counteracted the effects of dRib on GSH, MDA, 4-HNE, and lipid ROS levels, as well as cell death. Additionally, 2-mercaptoethanol (2-ME) treatment or xCT gene overexpression protected against dRib-induced changes. Moreover, transmission electron microscopy revealed dRib-induced mitochondrial shrinkage, decrease in cristae number, and outer membrane rupture. Furthermore, dRib treatment upregulated the expression of genes associated with ferroptosis, and downregulated xCT protein expression. The decrease in xCT protein caused by dRib was consistently observed even when treated with the protein synthesis inhibitor cycloheximide. However, treatment with the proteasome inhibitor MG132 reversed the dRib-induced decrease in xCT protein expression. Additionally, dRib increased xCT protein ubiquitination.Overall, dRib induces ferroptosis in RTECs by degrading xCT protein through ubiquitin-proteasome system (UPS), resulting in reduced intracellular cystine uptake. Therefore, targeting the regulation of system χc-through UPS could be a potential therapeutic approach for AKI and diabetic nephropathy.

P2X7 receptor inhibition ameliorates ubiquitin–proteasome system dysfunction associated with Alzheimer’s disease

BackgroundOver recent years, increasing evidence suggests a causal relationship between neurofibrillary tangles (NFTs) formation, the main histopathological hallmark of tauopathies, including Alzheimer’s disease (AD), and the ubiquitin–proteasome system (UPS) dysfunction detected in these patients. Nevertheless, the mechanisms underlying UPS failure and the factors involved remain poorly understood. Given that AD and tauopathies are associated with chronic neuroinflammation, here, we explore if ATP, one of the danger-associated molecules patterns (DAMPs) associated with neuroinflammation, impacts on AD-associated UPS dysfunction.MethodsTo evaluate if ATP may modulate the UPS via its selective P2X7 receptor, we combined in vitro and in vivo approaches using both pharmacological and genetic tools. We analyze postmortem samples from human AD patients and P301S mice, a mouse model that mimics pathology observed in AD patients, and those from the new transgenic mouse lines generated, such as P301S mice expressing the UPS reporter UbG76V-YFP or P301S deficient of P2X7R.ResultsWe describe for the first time that extracellular ATP-induced activation of the purinergic P2X7 receptor (P2X7R) downregulates the transcription of β5 and β1 proteasomal catalytic subunits via the PI3K/Akt/GSK3/Nfr2 pathway, leading to their deficient assembly into the 20S core proteasomal complex, resulting in a reduced proteasomal chymotrypsin-like and postglutamyl-like activities. Using UPS-reported mice (UbGFP mice), we identified neurons and microglial cells as the most sensitive cell linages to a P2X7R-mediated UPS regulation. In vivo pharmacological or genetic P2X7R blockade reverted the proteasomal impairment developed by P301S mice, which mimics that were detected in AD patients. Finally, the generation of P301S;UbGFP mice allowed us to identify those hippocampal cells more sensitive to UPS impairment and demonstrate that the pharmacological or genetic blockade of P2X7R promotes their survival.ConclusionsOur work demonstrates the sustained and aberrant activation of P2X7R caused by Tau-induced neuroinflammation contributes to the UPS dysfunction and subsequent neuronal death associated with AD, especially in the hippocampus.

A novel ubiquitin-proteasome system regulation of Sgf73/ataxin-7 that maintains the integrity of the coactivator SAGA in orchestrating transcription.

Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73's abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7's abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases.

Molecular mechanisms of cancer cachexia-related loss of skeletal muscle mass: data analysis from preclinical and clinical studies.

Cancer cachexia is a systemic hypoanabolic and catabolic syndrome that diminishes the quality of life of cancer patients, decreases the efficiency of therapeutic strategies and ultimately contributes to decrease their lifespan. The depletion of skeletal muscle compartment, which represents the primary site of protein loss during cancer cachexia, is of very poor prognostic in cancer patients. In this review, we provide an extensive and comparative analysis of the molecular mechanisms involved in the regulation of skeletal muscle mass in human cachectic cancer patients and in animal models of cancer cachexia. We summarize data from preclinical and clinical studies investigating how the protein turnover is regulated in cachectic skeletal muscle and question to what extent the transcriptional and translational capacities, as well as the proteolytic capacity (ubiquitin-proteasome system, autophagy-lysosome system and calpains) of skeletal muscle are involved in the cachectic syndrome in human and animals. We also wonder how regulatory mechanisms such as insulin/IGF1-AKT-mTOR pathway, endoplasmic reticulum stress and unfolded protein response, oxidative stress, inflammation (cytokines and downstream IL1ß/TNFα-NF-κB and IL6-JAK-STAT3 pathways), TGF-ß signalling pathways (myostatin/activin A-SMAD2/3 and BMP-SMAD1/5/8 pathways), as well as glucocorticoid signalling, modulate skeletal muscle proteostasis in cachectic cancer patients and animals. Finally, a brief description of the effects of various therapeutic strategies in preclinical models is also provided. Differences in the molecular and biochemical responses of skeletal muscle to cancer cachexia between human and animals (protein turnover rates, regulation of ubiquitin-proteasome system and myostatin/activin A-SMAD2/3 signalling pathways) are highlighted and discussed. Identifying the various and intertwined mechanisms that are deregulated during cancer cachexia and understanding why they are decontrolled will provide therapeutic targets for the treatment of skeletal muscle wasting in cancer patients.

The role of ubiquitination in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative and neuromuscular genetic disease caused by the expansion of a polyglutamine-encoding CAG tract in the androgen receptor (AR) gene. The AR is an important transcriptional regulator of the nuclear hormone receptor superfamily; its levels are regulated in many ways including by ubiquitin-dependent degradation. Ubiquitination is a post-translational modification (PTM) which plays a key role in both AR transcriptional activity and its degradation. Moreover, the ubiquitin-proteasome system (UPS) is a fundamental component of cellular functioning and has been implicated in diseases of protein misfolding and aggregation, including polyglutamine (polyQ) repeat expansion diseases such as Huntington’s disease and SBMA. In this review, we discuss the details of the UPS system, its functions and regulation, and the role of AR ubiquitination and UPS components in SBMA. We also discuss aspects of the UPS that may be manipulated for therapeutic effect in SBMA.

More Than Just Cleaning: Ubiquitin-Mediated Proteolysis in Fungal Pathogenesis.

Ubiquitin-proteasome mediated protein turnover is an important regulatory mechanism of cellular function in eukaryotes. Extensive studies have linked the ubiquitin-proteasome system (UPS) to human diseases, and an array of proteasome inhibitors have been successfully developed for cancer therapy. Although still an emerging field, research on UPS regulation of fungal development and virulence has been rapidly advancing and has generated considerable excitement in its potential as a target for novel drugs. In this review, we summarize UPS composition and regulatory function in pathogenic fungi, especially in stress responses, host adaption, and fungal pathogenesis. Emphasis will be given to UPS regulation of pathogenic factors that are important for fungal pathogenesis. We also discuss future potential therapeutic strategies for fungal infections based on targeting UPS pathways.

Dynamic Regulation of Histone Acetylation By Nuclear Proteolysis Controls Cell Cycle Gene Expression and Chromosome Integrity in Multiple Myeloma

Transcription factors are generally short-lived proteins that undergo active turnover. The dynamic interaction of transcription factors and co-regulators with promoters and enhancers allows cells to continuously adjust gene expression. Whereas the composition and binding of transcription factors at genomic sites is the focus of a widespread research effort, relatively little is known about how these complexes are being removed by the ubiquitin-proteasome system (UPS).Multiple myeloma (MM), the second most common hematopoietic malignancy, has become a model disease for drugs that interfere with the UPS through either blocking or facilitating protein elimination. The proteasome inhibitor Bortezomib, for instance, is used as first-line treatment in myeloma; yet, the process by which myeloma cells are killed by this drug is ill-defined. Since transcription factors are prime targets of proteasomal degradation, our research is focused on defining how proteolysis regulates transcriptional dynamics in this disease and determining the therapeutic relevance for treatment with proteasome inhibitors.Following proteasome inhibition in multiple myeloma cell lines, we performed chromatin-immunoprecipitation for histone H3 acetylation (K27) and multiple histone deacetylases and used next generation sequencing (ChIP-seq) to identify unique gene clusters that are actively regulated by the proteasome and quantify epigenetic changes in dependence of protein turnover. Our findings reveal that cell cycle genes, particularly subsets of genes involved in centromere formation and sister chromatid segregation during mitosis, are associated with nuclear protein turnover and are transcriptionally repressed by proteasome inhibition. Notably, proteasome inhibition increased the recruitment of specific histone deacetylases (HDACs) to the promoters of genes involved in centromere formation. We are investigating the mechanisms for UPS regulation of HDAC abundance at specific genomic locations. Moreover, data analysis of a panel of multiple myeloma patients shows that the expression levels of HDACs correlate with patient survival, specifically when treated with proteasome inhibitors. We are currently exploring how histone modifications and proteasome activity crosstalk in a therapeutically relevant manner in multiple myeloma.Finally, to determine whether epigenetic regulation of centromere stability might play a role in sensitivity or the development of resistance to proteasome inhibitors, we established and optimized long-term treatment of multiple myeloma cell lines with low doses of proteasome inhibitors. Remarkably, we observed reduced immunofluorescence staining for centromeric protein A (CENP-A) in multiple myeloma cells receiving drugs over an extended period, suggesting a loss of centromere integrity.This research project will contribute to our understanding of epigenetic and transcriptional dynamics in MM. With our focus on the continuously changing abundance of transcription factors and co-regulators at promoters of cell cycle genes, we seek to unlock new pathways for molecular therapy, as well as identify more specific targets for treatment compared to blunt proteasome inhibition. Elucidating the mechanisms of action and resistance to proteasome inhibition will help advance future MM therapy. DisclosuresLulla:ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees. Catic:Cancer Prevention and Research Institute of Texas: Research Funding; Ted Nash Long Life Foundation: Research Funding.

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.

The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analysed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the number of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response.

Dysregulation of the Ubiquitin Proteasome System in Human Malignancies: A Window for Therapeutic Intervention.

Simple SummaryThe ubiquitin proteasome system (UPS) governs the non-lysosomal degradation of oxidized, damaged, or misfolded proteins in eukaryotic cells. Dysregulation of the UPS results in loss of ability to maintain protein quality through proteolysis, and is closely related to the development of various malignancies and tumorigenesis. Here, we provide a comprehensive general overview on the regulation and roles of UPS and discuss the mechanisms linking dysregulated UPS to human malignancies. Inhibitors developed against components of the UPS, which include U.S. Food and Drug Administration FDA-approved and those currently undergoing clinical trials, are also presented in this review.The ubiquitin proteasome system (UPS) governs the non-lysosomal degradation of oxidized, damaged, or misfolded proteins in eukaryotic cells. This process is tightly regulated through the activation and transfer of polyubiquitin chains to target proteins which are then recognized and degraded by the 26S proteasome complex. The role of UPS is crucial in regulating protein levels through degradation to maintain fundamental cellular processes such as growth, division, signal transduction, and stress response. Dysregulation of the UPS, resulting in loss of ability to maintain protein quality through proteolysis, is closely related to the development of various malignancies and tumorigenesis. Here, we provide a comprehensive general overview on the regulation and roles of UPS and discuss functional links of dysregulated UPS in human malignancies. Inhibitors developed against components of the UPS, which include U.S. Food and Drug Administration FDA-approved and those currently undergoing clinical trials, are also presented in this review.

Adaptability of the ubiquitin-proteasome system to proteolytic and folding stressors.

Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.

TXLNB Signaling may not Intersect with Cardiac Proteotoxicity by CRYAB‐R120G

Aberrant protein folding/clearance in cardiomyocytes, via ubiquitin‐proteasome system (UPS) insufficiency, contributes to cardiomyopathy; however, our lab’s new data challenges this paradigm. Prior rodent studies show that UPS inhibition (pharmacologic and genetic) causes accumulation of misfolded/ubiquitinated proteins, decreased proteasomal activities, worse outcomes to cardiac stress, and premature death. Conversely, gain‐of‐function models enhancing UPS efficiency improve cardiac function.We discovered an exception to these associations while characterizing TXLNB, a muscle‐enriched protein with predominant perinuclear localization and unknown function. Surprisingly, despite severe cardiac UPS insufficiency, TXLNB knockout (TXLNB‐KO) mice exhibit normal cardiac function (Echo/EKG/LV‐Cath) up to 2 years‐old. In cardiomyocytes, TXLNB overexpression decreased ubiquitinated proteins and increased proteasome activity, whereas knockdown promotes proteasomal insufficiency. Similarly, hearts from young and old TXLNB‐KO mice show robust accumulation of ubiquitinated proteins (&gt;2‐fold) and decreased 26SB5 proteasome activity (~40%), similar to other UPS‐disruption models that show overt cardiac dysfunction (e.g. CRYAB‐R120G).Next, to test if cardiotoxic UPS dysfunction intersects with TXLNB actions, TXLNB‐KO mice were crossed to CRYAB‐R120G transgenic mice and heart functions measured in mixed‐sex cohorts for one year. Our data show CRYAB mice present with characteristic bi‐ventricular hypertrophy that slowly progresses to heart failure (EF = 56% at 36 weeks age, n=28), with intracardiac thrombi, pericardial effusion, pulmonary edema, and early death (median survival=41.8 weeks). Combined TXLNB‐KO / CRYAB mice trended to worse cardiac function (EF =46% at 36 weeks age, n=16, p=0.057 compared to CRYAB only), however end‐stage heart failure remained unchanged (median survival=42.7).Next, to test if TXLNB overexpression could improve CRYAB‐induced proteotoxicity, we created an AAV2/9 virus that expresses TXLNB under control of cardiac Troponin T promoter (cTnT), capable of 6‐fold overexpression in hearts. Wild‐type and CRYAB mice at 3 weeks old were injected via jugular vein with AAV2/9 cTnT‐TXLNB (n=6) or cTNT‐GFP (n=6) virus and monitored until 25 weeks age. EKG, Echocardiography, and post‐mortem gravimetric results show that TXLNB overexpression does not reduce biventricular hypertrophy, or improve cardiac functions caused by CRYAB‐R120G. Ongoing work is evaluating if TXLNB overexpression restores any molecular deficiencies caused by CRYAB‐R120G (proteasome insufficiency, ubiquitinated protein load, oxidized proteins, etc).Together, these findings suggest that TXLNB regulation of the UPS and cardiomyopathy from proteotoxic stress exist separately, and the understood relationship between UPS insufficiency and cardiomyopathy remains unclear. We think the prominent perinuclear location of TXLNB indicates a role in UPS regulation at microtubule centers and may associate with redistributed centrosomal proteins in cardiomyocytes. Ongoing work will address the “where” and/or the “what” (regarding accumulated ubiquitinated proteins) that dictate pathological/protective responses.Support or Funding Information19POST34380640 to JMM and HL144717, HL148796 to RLB

An F-Box Protein, Mdm30, Interacts with TREX Subunit Sub2 To Regulate Cellular Abundance Cotranscriptionally in Orchestrating mRNA Export Independently of Splicing and Mitochondrial Function.

Although an F-box protein, Mdm30, is found to regulate ubiquitylation of the Sub2 component of TREX (transcription-export) complex for proteasomal degradation in stimulation of mRNA export, it remains unknown whether such ubiquitin-proteasome system (UPS) regulation of Sub2 occurs cotranscriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in the absence of Mdm30 alters mRNA export via splicing defects of export factors and/or mitochondrial dynamics/function, since Sub2 controls mRNA splicing and Mdm30 regulates mitochondrial aggregation. Here, we show that Mdm30 interacts with Sub2, and temporary shutdown of Mdm30 enhances Sub2's abundance and impairs mRNA export. Likewise, Sub2's abundance is increased following transcriptional inhibition. These results support Mdm30's direct role in regulation of Sub2's cellular abundance in a transcription-dependent manner. Consistently, the chromatin-bound Sub2 level is increased in the absence of Mdm30. Further, we find that Mdm30 does not facilitate splicing of export factors. Moreover, Mdm30 does not have a dramatic effect on mitochondrial respiration/function, and mRNA export occurs in the absence of Fzo1, which is required for mitochondrial dynamics/respiration. Collective results reveal that Mdm30 interacts with Sub2 for proteasomal degradation in a transcription-dependent manner to promote mRNA export independently of splicing or mitochondrial function, thus advancing our understanding of mRNA export.

Emergence of Members of TRAF and DUB of Ubiquitin Proteasome System in the Regulation of Hypertrophic Cardiomyopathy.

The ubiquitin proteasome system (UPS) plays an imperative role in many critical cellular processes, frequently by mediating the selective degradation of misfolded and damaged proteins and also by playing a non-degradative role especially important as in many signaling pathways. Over the last three decades, accumulated evidence indicated that UPS proteins are primal modulators of cell cycle progression, DNA replication, and repair, transcription, immune responses, and apoptosis. Comparatively, latest studies have demonstrated a substantial complexity by the UPS regulation in the heart. In addition, various UPS proteins especially ubiquitin ligases and proteasome have been identified to play a significant role in the cardiac development and dynamic physiology of cardiac pathologies such as ischemia/reperfusion injury, hypertrophy, and heart failure. However, our understanding of the contribution of UPS dysfunction in the plausible development of cardiac pathophysiology and the complete list of UPS proteins regulating these afflictions is still in infancy. The recent emergence of the roles of TNF receptor-associated factor (TRAFs) and deubiquitinating enzymes (DUBs) superfamily in hypertrophic cardiomyopathy has enhanced our knowledge. In this review, we have mainly compiled the TRAF superfamily of E3 ligases and few DUBs proteins with other well-documented E3 ligases such as MDM2, MuRF-1, Atrogin-I, and TRIM 32 that are specific to myocardial hypertrophy. In this review, we also aim to highlight their expression profile following physiological and pathological stimulation leading to the onset of hypertrophic phenotype in the heart that can serve as biomarkers and the opportunity for the development of novel therapies.

Regulation of an Evolutionarily Conserved RNA Polymerase II‐Associated Factor 1 (Paf1) Involved in Pancreatic Oncogenesis

The multifunctional RNA polymerase II‐associated factor (Paf1) is highly conserved from yeast to humans and plays an important role in regulation of transcription and its associated processes. Hence, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Recent studies found that Paf1 (also known as PD2 or pancreatic differentiation 2) is upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In light of this, we have tested here the idea that the ubiquitin‐proteasome system (UPS) regulates the cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1's abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well‐differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Thus, our results reveal UPS regulation of Paf1 and suggest downregulation of UPS in elevating Paf1's abundance in poorly differentiated cancers. Further, we find that misregulation of Paf1's abundance alters transcription. Collectively, our results demonstrate that Paf1 is regulated by UPS to control transcription, thus providing a new regulatory mechanism of gene expression.Support or Funding InformationNIH‐2R15GM088798‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ubiquitin-Proteasome System Regulation of an Evolutionarily Conserved RNA Polymerase II-Associated Factor 1 Involved in Pancreatic Oncogenesis.

The evolutionarily conserved RNA polymerase II-associated factor 1 (Paf1) from yeast to humans regulates transcription and associated processes, and thus, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Indeed, Paf1 (also known as PD2 or pancreatic differentiation 2) is found to be upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In light of this, we have tested here the idea that the ubiquitin-proteasome system (UPS) regulates the cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1's abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well-differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Collectively, our results reveal UPS regulation of Paf1 and suggest downregulation of UPS in elevating Paf1's abundance in poorly differentiated cancers.

Correction for Sen et al., "Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation".

Correction for Sen et al., "Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation".