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Background & Aims Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. Methods We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. Results The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2–hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. Conclusions The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans., Graphical abstract

Cells have different sets of molecules for performing an array of physiological functions. Nucleic acids have stored and carried the information throughout evolution, whereas proteins have been attributed to performing most of the cellular functions. To perform these functions, proteins need to have a unique conformation and a definite lifespan. These attributes are achieved by a highly coordinated protein quality control (PQC) system comprising chaperones to fold the proteins in a proper three-dimensional structure, ubiquitin-proteasome system for selective degradation of proteins, and autophagy for bulk clearance of cell debris. Many kinds of stresses and perturbations may lead to the weakening of these protective cellular machinery, leading to the unfolding and aggregation of cellular proteins and the occurrence of numerous pathological conditions. However, modulating the expression and functional efficiency of molecular chaperones, E3 ubiquitin ligases, and autophagic proteins may diminish cellular proteotoxic load and mitigate various pathological effects. Natural medicine and small molecule-based therapies have been well-documented for their effectiveness in modulating these pathways and reestablishing the lost proteostasis inside the cells to combat disease conditions. The present article summarizes various similar reports and highlights the importance of the molecules obtained from natural sources in disease therapeutics., Graphical abstract Extra-/intracellular stresses challenge cellular health throughout life. Small natural molecules modulate the functioning of protein quality control components to maintain proteins' native structures and preserve cells’ proteome under healthy and stressful conditions.Image 1

Interferon Regulatory Factors (IRFs) are key regulators of immunity, cell survival and apoptosis. IRF transcriptional activity and subcellular localization are tightly regulated by posttranscriptional modifications including phosphorylation. The IκB kinase family member IKK-ε is essential in regulating antiviral innate immunity mediated by IRFs but is now also recognized as an oncoprotein amplified and overexpressed in breast cancer cell lines and patient-derived tumors. In the present study, we report that the tumor suppressor IRF-1 is a specific target of IKK-ε in breast cancer cells. IKK-ε-mediated phosphorylation of IRF-1 dramatically decreases IRF-1 protein stability, accelerating IRF-1 degradation and quenching IRF-1 transcriptional activity. Chemical inhibition of IKK-ε activity, fully restores IRF-1 levels and function and positively correlates with inhibition of cell growth and proliferation of breast cancer cells. By using a breast cancer cell line stably expressing a dominant negative version of IRF-1 we were able to demonstrate that IKK-ε preferentially exerts its oncogenic potential in breast cancer through the regulation of IRF-1 and point to the IKK-ε-mediated phosphorylation of IRF-1 as a therapeutic target to overcome IKK-ε-mediated tumorigenesis.

Ubiquitin specific peptidase 28 (USP28) is closely associated to the occurrence and development of various malignancies, and thus has been validated as a promising therapeutic target for cancer therapy. To date, only few USP28 inhibitors with moderate inhibitory activity have been reported, highly potent and selective USP28 inhibitors with new chemotypes remain to be discovered for pathologically investigating the roles of deubiquitinase. In this current study, we reported the synthesis and biological evaluation of new [1,2,3]triazolo[4,5-d]pyrimidine derivatives as potent USP28 inhibitors. Especially, compound 19 potently inhibited USP28 (IC50 = 1.10 ± 0.02 μmol/L, Kd = 40 nmol/L), showing selectivity over USP7 and LSD1 (IC50 > 100 μmol/L). Compound 19 was cellularly engaged to USP28 in gastric cancer cells. Compound 19 reversibly bound to USP28 and directly affected its protein levels, thus inhibiting the proliferation, cell cycle at S phase, and epithelial-mesenchymal transition (EMT) progression in gastric cancer cell lines. Docking studies were performed to rationalize the potency of compound 19. Collectively, compound 19 could serve as a new tool compound for the development of new USP28 inhibitors for exploring the roles of deubiquitinase in cancers., Graphical abstract A new series of [1,2,3]triazolo[4,5-d]pyrimidine derivatives were identified to inhibit USP28. Compound 19 potently inhibited USP28 with the IC50 and Kd values of 1.1 μmol/L and 40 nmol/L, respectively.Image 1

The deubiquitinating enzyme USP37 is known to contribute to timely onset of S phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase 1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates checkpoint kinase 1, promoting its stability. Together, our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.

The contribution of the Ubiquitin-Proteasome System (UPS) to mitophagy has been largely attributed to the E3 ubiquitin ligase Parkin. Here we show that in response to the oxidative stress associated with hypoxia or the hypoxia mimic CoCl2, the damaged and fragmented mitochondria are removed by Parkin-independent mitophagy. Mitochondria isolated from hypoxia or CoCl2-treated cells exhibited extensive ubiquitination, predominantly Lysine 48-linked and involves the degradation of key mitochondrial proteins such as the mitofusins MFN1/2, or the import channel component TOM20. Reflecting the critical role of mitochondrial protein degradation, proteasome inhibition blocked CoCl2-induced mitophagy. The five conserved ubiquitin-binding autophagy receptors (p62, NDP52, Optineurin, NBR1, TAX1BP1) were dispensable for the ensuing mitophagy, suggesting that the mitophagy step itself was independent of ubiquitination. Instead, the expression of two ubiquitin-independent mitophagy receptor proteins BNIP3 and NIX was induced by hypoxia or CoCl2-treatment followed by their recruitment to the oxidation-damaged mitochondria. By employing BNIP3/NIX double knockout and DRP1-null cell lines, we confirmed that mitochondrial clearance relies on DRP1-dependent mitochondrial fragmentation and BNIP3/NIX-mediated mitophagy. General antioxidants such as N-Acetyl Cysteine (NAC) or the mitochondria-specific Mitoquinone prevented HIF-1α stabilization, ameliorated hypoxia-related mitochondrial oxidative stress, and suppressed mitophagy. We conclude that the UPS and receptor-mediated autophagy converge to eliminate oxidation-damaged mitochondria., Graphical abstract Sulkshane P. et al. show that oxidation-induced mitophagy entails two steps: degradation of outer mitochondrial membrane proteins in a ubiquitin-proteasome-dependent manner followed by ubiquitin-independent mitophagy.Image 1, Highlights • Mitochondria-derived ROS contributes to HIF-1α stabilization during hypoxia. • Oxidation-induced mitophagy entails ubiquitin-dependent and -independent steps. • PINK1/Parkin & Ub-binding receptors are dispensable for oxidation-induced mitophagy. • DRP1-dependent fragmentation facilitates oxidation-induced mitophagy. • BNIP3/NIX partake in hypoxia-induced mitophagy, independent of mitochondrial ubiquitination.

The hepatic endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) are mixed-function oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance. P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover. Such P450 proteolytic turnover occurs through a process known as ER-associated degradation (ERAD) that involves ubiquitin-dependent proteasomal degradation (UPD) and/or autophagic-lysosomal degradation (ALD). Herein, on the basis of available literature reports and our own recent findings of in vitro as well as in vivo experimental studies, we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance. We specifically (i) describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibody pathogenesis in drug-induced acute hypersensitivity reactions and liver injury, or viral hepatitis; (ii) discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates; and (iii) detail the pathophysiological consequences of disrupted P450 ERAD, contributing to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) under certain synergistic cellular conditions., Graphical abstract In this article, we discuss the physiological, therapeutic and pathophysiological relevance of cytochrome P450 protein degradation. Employing mice with genetic ablation of CHIP and gp78/AMFR E3 ubiquitin-ligases that stabilize hepatic CYP2E1, we document why this stabilization not always leads to non-alcoholic fatty liver disease: It requires concomitantly enhanced hepatic lipogenesis.Image 1

Marafiviruses are capable of persistent infection in a range of plants that have importance to the agriculture and biofuel industries. Although the genomes of a few of these viruses have been studied in-depth, the composition and processing of the polyproteins produced from their main ORFs have not. The Marafivirus polyprotein consists of essential proteins that form the viral replicase, as well as structural proteins for virus assembly. It has been proposed that Marafiviruses code for cysteine proteases within their polyproteins, which act as endopeptidases to autocatalytically cleave the polyprotein into functional domains. Furthermore, it has also been suggested that Marafivirus endopeptidases may have deubiquitinating activity, which has been shown to enhance viral replication by downregulating viral protein degradation by the ubiquitin (Ub) proteasomal pathway as well as tampering with cell signaling associated with innate antiviral responses in other positive-sense ssRNA viruses. Here, we provide the first evidence of cysteine proteases from six different Marafiviruses that harbor deubiquitinating activity and reveal intragenus differences toward Ub linkage types. We also examine the structural basis of the endopeptidase/deubiquitinase from the Marafivirus type member, maize rayado fino virus. Structures of the enzyme alone and bound to Ub reveal marked structural rearrangements that occur upon binding of Ub and provide insights into substrate specificity and differences that set it apart from other viral cysteine proteases.

Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment (e.g., nutrient fluctuations or different stresses). At the heart of this mechanism lies a network of proteins that include the arrestin?related trafficking adaptors (ARTs) which link the ubiquitin ligase Rsp5 to nutrient transporters and endocytic factors. Transporter conformational changes, as well as dynamic interactions between its cytosolic termini/loops and with lipids of the plasma membrane, are also critical during the endocytic process. Here, we review the current knowledge and recent findings on the molecular mechanisms involved in nutrient transporter endocytosis, both in the budding yeast Saccharomyces cerevisiae and in some species of the filamentous fungus Aspergillus. We elaborate on the physiological importance of tightly regulated endocytosis for cellular fitness under dynamic conditions found in nature and highlight how further understanding and engineering of this process is essential to maximize titer, rate and yield (TRY)-values of engineered cell factories in industrial biotechnological processes., We are grateful to Dr. George Diallinas and Dr. Christos Gournas for their suggestions and critical reading of this manuscript. We thank Dr. Jack Pronk for his fruitful suggestions. Work at CBMA was supported by the ‘‘Contrato-Programa” UIDB/04050/2020 funded by Portuguese national funds through the FCT I.P. BA and RA acknowledge FCT for the PD/BD/135208/2017 and PD/BD/113813/2015 PhD grants, respectively., info:eu-repo/semantics/publishedVersion

Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub–substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate–Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.

Access of molecular oxygen to the respiratory electron transport chain at the mitochondria costs in the generation of reactive oxygen-derived species (ROS). ROS induces progressive damage to macromolecules in all living cells, hence, rapid defense mechanisms to maintain cellular redox homeostasis are vital. NEDD8/Rub1 is a highly conserved ubiquitin-like modifier that has recently been identified as a key regulator of cellular redox homeostasis. In this review, I will present NEDD8/Rub1, its modification cascade of enzymes, substrates and hydrolases. After introduction, I will show that the NEDD8/Rub1 pathway is linked with mitochondria physiology, namely, oxidative stress. In the rest of the review, I will approach the Ascomycota phylum of the kingdom fungi instrumentally, to present existing links between NEDD8/Rub1 vitality and the aerobic lifestyle of model species belonging to three subphyla: Saccharomycotina (S. cerevisiae and C. albicans), Pezizomycotina (A. nidulans and N. crassa), and Taphrinomycotina (S. pombe)., Graphical abstract Image 1, Highlights • NEDD8/Rub1 is a key regulator of cellular redox homeostasis. • Ascomycota species that produce mitochondria-derived ROS during glycolysis require NEDD8/Rub1for viability. • NEDD8/Rub1 essentiality correlates with the existence of NEDP1 in the organism genome.

Genetic porphyrias comprise eight diseases caused by defects in the heme biosynthetic pathway that lead to accumulation of heme precursors. Consequences of porphyria include photosensitivity, liver damage and increased risk of hepatocellular carcinoma, and neurovisceral involvement, including seizures. Fluorescent porphyrins that include protoporphyrin-IX, uroporphyrin and coproporphyrin, are photo-reactive; they absorb light energy and are excited to high-energy singlet and triplet states. Decay of the porphyrin excited to ground state releases energy and generates singlet oxygen. Porphyrin-induced oxidative stress is thought to be the major mechanism of porphyrin-mediated tissue damage. Although this explains the acute photosensitivity in most porphyrias, light-induced porphyrin-mediated oxidative stress does not account for the effect of porphyrins on internal organs. Recent findings demonstrate the unique role of fluorescent porphyrins in causing subcellular compartment-selective protein aggregation. Porphyrin-mediated protein aggregation associates with nuclear deformation, cytoplasmic vacuole formation and endoplasmic reticulum dilation. Porphyrin-triggered proteotoxicity is compounded by inhibition of the proteasome due to aggregation of some of its subunits. The ensuing disruption in proteostasis also manifests in cell cycle arrest coupled with aggregation of cell proliferation-related proteins, including PCNA, cdk4 and cyclin B1. Porphyrins bind to native proteins and, in presence of light and oxygen, oxidize several amino acids, particularly methionine. Noncovalent interaction of oxidized proteins with porphyrins leads to formation of protein aggregates. In internal organs, particularly the liver, light-independent porphyrin-mediated protein aggregation occurs after secondary triggers of oxidative stress. Thus, porphyrin-induced protein aggregation provides a novel mechanism for external and internal tissue damage in porphyrias that involve fluorescent porphyrin accumulation. Keywords: Porphyria, Oxidative Stress, Protein Aggregation, Phototoxicity

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blocking therapy has become a major pillar of cancer immunotherapy. Compared with antibodies targeting, small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed. Here we identified berberine (BBR), a proven anti-inflammation drug, as a negative regulator of PD-L1 from a set of traditional Chinese medicine (TCM) chemical monomers. BBR enhanced the sensitivity of tumour cells to co-cultured T-cells by decreasing the level of PD-L1 in cancer cells. In addition, BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumor-infiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). BBR triggered PD-L1 degradation through ubiquitin (Ub)/proteasome-dependent pathway. Remarkably, BBR selectively bound to the glutamic acid 76 of constitutive photomorphogenic-9 signalosome 5 (CSN5) and inhibited PD-1/PD-L1 axis through its deubiquitination activity, resulting in ubiquitination and degradation of PD-L1. Our data reveals a previously unrecognized antitumor mechanism of BBR, suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment., Graphical abstract Berberine diminishes the expression of programmed cell death ligand-1 and promotes antitumor immunity via inhibiting the deubiquitination activity of COP9 signalosome 5 (CSN5) in non-small cell lung cancer.Image 1

Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.

The ubiquitin (Ub)-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy-in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors. Moreover, the largest proteasomal receptor, Rpn1, contains one known binding site for polyubiquitin and shuttle proteins, although several studies have recently proposed the existence of an additional uncharacterized site. Here, using a combination of NMR spectroscopy, photocrosslinking, mass spectrometry, and mutagenesis, we show that Rpn1 does indeed contain another recognition site that exhibits affinities and binding preferences for polyubiquitin and Ub-like signals comparable to those of the known binding site in Rpn1. Surprisingly, this novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality. We identified a stretch of adjacent helices as the location of this previously uncharacterized binding site, whose spatial proximity and similar properties to the known binding site in Rpn1 suggest the possibility of multivalent signal recognition across the solvent-exposed surface of Rpn1. These findings offer new mechanistic insights into signal recognition processes that are at the core of the Ub-proteasome system.

Oxysterols are bioactive lipids that act as regulators of lipid metabolism, inflammation, cell viability and are involved in several diseases, including atherosclerosis. Mounting evidence linked the atherosclerosis to endothelium dysfunction; in fact, the endothelium regulates the vascular system with roles in processes such as hemostasis, cell cholesterol, hormone trafficking, signal transduction and inflammation. Several papers shed light the ability of oxysterols to induce apoptosis in different cell lines including endothelial cells. Apoptotic endothelial cell and endothelial denudation may constitute a critical step in the transition to plaque erosion and vessel thrombosis, so preventing the endothelial damaged has garnered considerable attention as a novel means of treating atherosclerosis. Endoplasmic reticulum (ER) is the site where the proteins are synthetized and folded and is necessary for most cellular activity; perturbations of ER homeostasis leads to a condition known as endoplasmic reticulum stress. This condition evokes the unfolded protein response (UPR) an adaptive pathway that aims to restore ER homeostasis. Mounting evidence suggests that chronic activation of UPR leads to cell dysfunction and death and recently has been implicated in pathogenesis of endothelial dysfunction. Autophagy is an essential catabolic mechanism that delivers misfolded proteins and damaged organelles to the lysosome for degradation, maintaining basal levels of autophagic activity it is critical for cell survival. Several evidence suggests that persistent ER stress often results in stimulation of autophagic activities, likely as a compensatory mechanism to relieve ER stress and consequently cell death. In this review, we summarize evidence for the effect of oxysterols on endothelial cells, especially focusing on oxysterols-mediated induction of endoplasmic reticulum stress., Graphical abstract fx1, Highlights • Endothelial cells dysfunction is critical in the process of atherothrombosis. • Endoplasmic reticulum stress is a key component in endothelial cell dysfunction. • Oxysterols are oxidation products of cholesterol found in atherosclerosis lesions. • Oxysterols are potential modulators of endoplasmic reticulum stress.

Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset., Graphical abstract fx1

Signaling cascades respond to specific inputs, but also require active interventions to be maintained in their basal/inactive levels in the absence of the activating signal(s). In a screen to search for protein quality control components required for wild-type tolerance to oxidative stress in fission yeast, we have isolated eight gene deletions conferring resistance not only to H2O2 but also to caffeine. We show that dual resistance acquisition is totally or partially dependent on the transcription factor Pap1. Some gene products, such as the ribosomal-ubiquitin fusion protein Ubi1, the E2 conjugating enzyme Ubc2 or the E3 ligase Ubr1, participate in basal ubiquitin labeling of Pap1, and others, such as Rpt4, are non-essential constituents of the proteasome. We demonstrate here that basal nucleo-cytoplasmic shuttling of Pap1, occurring even in the absence of stress, is sufficient for the interaction of the transcription factor with nuclear Ubr1, and we identify a 30 amino acids peptide in Pap1 as the degron for this important E3 ligase. The isolated gene deletions increase only moderately the concentration of the transcription factor, but it is sufficient to enhance basal tolerance to stress, probably by disturbing the inactive stage of this signaling cascade. This work is supported by the Ministerio de Economía y Competitividad (Spain), PLAN E and FEDER (BFU2015-68350-P and PGC2018-093920 to E.H) and by Unidad de Excelencia María de Maeztu (MDM-2014-0370). The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539). E. H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain).

Enzymatic and non-enzymatic oxidation of unsaturated fatty acids gives rise to reactive species that covalently modify nucleophilic residues within redox sensitive protein sensors in a process called lipoxidation. This triggers adaptive signaling pathways that ultimately lead to increased resistance to stress. In this graphical review, we will provide an overview of pathways affected by protein lipoxidation and the key signaling proteins being altered, focusing on the KEAP1-NRF2 and heat shock response pathways. We review the mechanisms by which lipid peroxidation products can serve as second messengers and evoke cellular responses via covalent modification of key sensors of altered cellular environment, ultimately leading to adaptation to stress., Graphical abstract fx1

We have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitin chains containing both Lys63-linked and Met1-linked ubiquitin (Ub) oligomers, some of which are attached covalently to Interleukin Receptor Associated kinase 1. Here we show that Lys63/Met1-Ub hybrids are also formed rapidly when the TNFR1/TRADD, TLR3/TRIF- and NOD1/RIP2-signaling networks are activated, some of which are attached covalently to Receptor-Interacting Protein 1 (TNFR1 pathway) or Receptor-Interacting Protein 2 (NOD1 pathway). These observations suggest that the formation of Lys63/Met1-Ub hybrids are of general significance for the regulation of innate immune signaling systems, and their potential roles in vivo are discussed. We also report that TNFα induces the attachment of Met1-linked Ub chains directly to TNF receptor 1, which do not seem to be attached covalently to Lys63-linked or other types of ubiquitin chain., Highlights • Ubiquitin chains containing both Lys63 and Met1 linkages are commonly formed during innate immune signaling. • Lys63/Met1-hybrid chains become attached to RIP1 and RIP2 in the TNFR1 and NOD1 signaling networks, respectively. • Potential advantages of Lys63/Met1-hybrids over separate ubiquitin chains are proposed. • Met1-linked ubiquitin is attached to TNFR1 without formation of a hybrid ubiquitin chain.

Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.

TRAF3 has diverse signaling functions, which vary by cell type. Uniquely in B lymphocytes, TRAF3 inhibits homeostatic survival. Highlighting the role of TRAF3 as a tumor suppressor, loss-of-function TRAF3 mutations are associated with human B-cell malignancies, while B-cell-specific deletion of TRAF3 in mice leads to autoimmunity and lymphoma development. The role of TRAF3 in inhibiting noncanonical NF-κB activation, CD40 and BAFF-R signaling to B cells is well documented. In contrast, TRAF3 enhances many T-cell effector functions, through associating with and enhancing signaling by the T-cell receptor (TCR)-CD28 complex. The present study was designed to determine the role of TRAF3 in signaling via the B-cell antigen receptor (BCR). The BCR is crucial for antigen recognition, survival, proliferation, and antibody production, and defects in BCR signaling can promote abnormal survival of malignant B cells. Here, we show that TRAF3 is associated with both CD79B and the BCR-activated kinases Syk and Btk following BCR stimulation. BCR-induced phosphorylation of Syk and additional downstream kinases was increased in TRAF3−/− B cells, with regulation observed in both follicular and marginal zone B-cell subsets. BCR stimulation of TRAF3−/− B cells resulted in increased surface expression of MHC-II, CD80, and CD86 molecules. Interestingly, increased survival of TRAF3−/− primary B cells was resistant to inhibition of Btk, while TRAF3-deficient malignant B-cell lines showed enhanced sensitivity. TRAF3 serves to restrain normal and malignant BCR signaling, with important implications for its role in normal B-cell biology and abnormal survival of malignant B cells.

The voltage-gated potassium channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (IKv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 channel function under PKC activation conditions.

RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-β (TGF-β) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-β signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-β signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-β. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-β pathway activation., Graphical abstract, Highlights • Identification of the endogenous RNF111 ubiquitylated substrates. • A new powerful method to profile protein ubiquitylation using pan UB nanobody. • Lysine K343 as a target for RNF111 ubiquitylation. • SKI and SKIL are the only identified RNF111 degradative targets in TGF-β signaling., In Brief In this study, we aimed to identify exhaustively the substrates of the E3 ubiquitin ligase RNF111 that activates TGF-β signaling. We performed quantitative ubiquitylome comparison of parental U2OS cells with CRISPR modified clones that express a truncated RNF111 devoid of RING domain using two approaches based on the enrichment of ubiquitylated proteins. Integrative proteomics comparison of ubiquitylome and proteome identifies SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 upon TGF-β stimulation.

Cullin RING E3 ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs), and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contributes to polyubiquitylation but does not alter the sites of CKB ubiquitylation. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high-affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates., Graphical Abstract, Highlights • ARIH2 is required for ASB9CRL to polyubiquitylate 4/18 lysines on one creatine kinase subunit. • HDX-MS reveals long-range allosteric opening of a cleft in CUL5 where the ARIH2 RBRL binds. • HDX-MS reveals that neddylation of CUL5 alters the RBX2 conformation away from binding the E2∼NEDD8. • HDX-MS reveals opening of the ARIH2 active site upon binding CUL5, thus releasing its autoinhibition., In Brief Hydrogen-deuterium exchange mass spectrometry (HDX-MS) reveals how a CUL5 E3 ligase is activated for polyubiquitylation of its substrate.

Interferon-stimulated gene product 15 (ISG15) is a key component of host responses to microbial infection. Despite having been known for four decades, grasping the functions and features of ISG15 has been a slow and elusive process. Substantial work over the past two decades has greatly enhanced this understanding, revealing the complex and variable nature of this protein. This has unveiled multiple mechanisms of action that are only now beginning to be understood. In addition, it has uncovered diversity not only between how ISG15 affects different pathogens but also between the function and structure of ISG15 itself between different host species. Here we review the complexity of ISG15 within the context of viral infection, focusing primarily on its antiviral function and the mechanisms viruses employ to thwart its effects. We highlight what is known regarding the impact of ISG15 sequence and structural diversity on these interactions and discuss the aspects presenting the next frontier toward elucidating a more complete picture of ISG15 function., Graphical abstract Unlabelled Image, Highlights • ISG15 is one of the most highly upregulated IFN-stimulated proteins. • ISG15 is involved in numerous pathways with multiple mechanisms of action. • Viruses encode specific countermeasures to combat ISG15 function. • ISG15 possesses interspecies sequence diversity impacting structure, function, and viral interactions. • Key advances have been made; much work remains to grasp the complexity of ISG15.

Cycles of Cdc53/Cullin1 rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skp1-Cullin1-F-box protein) complexes from self-destruction and play an important role in mediating the ubiquitination of key protein substrates involved in cell cycle progression, development, and survival. Cul1 rubylation is balanced by the COP9 signalosome (CSN), a multi-subunit derubylase that shows 1:1 paralogy to the 26S proteasome lid. The turnover of SCF substrates and their relevance to various diseases is well studied, yet, the extent by which environmental perturbations influence Cul1 rubylation/derubylation cycles per se is still unclear. In this study, we show that the level of cellular oxidation serves as a molecular switch, determining Cullin1 rubylation/derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpn11 that exhibits accumulated levels of Cullin1-Rub1 conjugates, a characteristic phenotype of csn mutants. By dissecting between distinct phenotypes of rpn11 mutants, proteasome and mitochondria dysfunction, we were able to recognize the high reactive oxygen species (ROS) production during the transition of cells into mitochondrial respiration, as a checkpoint of Cullin1 rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis., Graphical abstract fx1, Highlights • Cullin rubylation status inversely correlates with the cellular redox state. • The yeast diauxic shift blocks cullin rubylation in a reversible manner. • Cullin rubylation status is determined by the oxidation state of individual cells. • The activity of rubylation cascade enzymes is perturbed by high ROS. • The loss of cullin rubylation correlates with the appearance of free Rub1 molecules.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a ‘proteostasis network’ and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge – the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress., Highlights • The proteostasis network is perturbed in neurological diseases. • UPR, UPS and autophagy are adaptive responses to maintain cell function. • Adaptive responses induced by preconditioning could be used for therapeutics.

In the last decade, the ubiquitin-proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin-RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs.

Modification of proteins with ubiquitin (Ub) occurs through a variety of topologically distinct Ub linkages, including Ube2W-mediated monoubiquitylation of N-terminal alpha amines to generate peptide-linked linear mono-Ub fusions. Protein ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs), many of which show striking preference for particular Ub linkage types. Here, we have screened for DUBs that preferentially cleave N-terminal Ub from protein substrates but do not act on Ub homopolymers. We show that members of the Ub C-terminal hydrolase (UCH) family of DUBs demonstrate this preference for N-terminal deubiquitylating activity as they are capable of cleaving N-terminal Ub from SUMO2 and Ube2W, while displaying no activity against any of the eight Ub linkage types. Surprisingly, this ability to cleave Ub from SUMO2 was 100 times more efficient for UCH-L3 when we deleted the unstructured N-terminus of SUMO2, demonstrating that UCH enzymes can cleave Ub from structured proteins. However, UCH-L3 could also cleave chemically synthesized isopeptide-linked Ub from lysine 11 (K11) of SUMO2 with similar efficiency, demonstrating that UCH DUB activity is not limited to peptide-linked Ub. These findings advance our understanding of the specificity of the UCH family of DUBs, which are strongly implicated in cancer and neurodegeneration but whose substrate preference has remained unclear. In addition, our findings suggest that the reversal of Ube2W-mediated N-terminal ubiquitylation may be one physiological role of UCH DUBs in vivo., The substrate preference of ubiquitin C-terminal hydrolase (UCH) deubiquitylating enzymes (DUBs) has been unclear. Here we report that UCH family DUBs efficiently cleave both isopeptide- and peptide-linked ubiquitin from substrates, without processing simple Ub–Ub polymers of any linkage type.

Highlights • 6-Mercaptopurine and 6-thioguanine are competitive inhibitors of SARS-CoV and MERS-CoV papain-like proteases. • Mycophenolic acid can noncompetitively inhibit papain-like protease of MERS-CoV, while cannot inhibit that of SARS-CoV. • 6-Mercaptopurine/6-thioguanine and mycophenolic acid synergistically inhibit MERS-CoV papain-like protease. • The three drugs with inhibitory effect for MERS-CoV papain-like protease can be used as lead compounds for drug development., Middle East respiratory syndrome coronavirus (MERS-CoV) is a new highly pathogenic human coronaviruses that emerged in Jeddah and Saudi Arabia and has quickly spread to other countries in Middle East, Europe and North Africa since 2012. Up to 17 December 2014, it has infected at least 938 people with a fatality rate of about 36% globally. This has resulted in an urgent need to identify antiviral drugs that are active against MERS-CoV. The papain-like protease (PLpro) of MERS-CoV represents an important antiviral target as it is not only essential for viral maturation, but also antagonizes interferon stimulation of the host via its deubiquitination activity. Here, we report the discovery that two SARS-CoV PLpro inhibitors, 6-mercaptopurine (6MP) and 6-thioguanine (6TG), as well as the immunosuppressive drug mycophenolic acid, are able to inhibit MERS-CoV PLpro. Their inhibition mechanisms and mutually binding synergistic effect were also investigated. Our results identify for the first time three inhibitors targeting MERS-CoV PLpro and these can now be used as lead compounds for further antiviral drug development.

Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box) motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.

The multi-domain non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome, with an average molecular mass of about 200 kD. Nsp3 is an essential component of the replication/transcription complex. It comprises various domains, the organization of which differs between CoV genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known CoVs: the ubiquitin-like domain 1 (Ubl1), the Glu-rich acidic domain (also called “hypervariable region”), a macrodomain (also named “X domain”), the ubiquitin-like domain 2 (Ubl2), the papain-like protease 2 (PL2pro), the Nsp3 ectodomain (3Ecto, also called “zinc-finger domain”), as well as the domains Y1 and CoV-Y of unknown functions. In addition, the two transmembrane regions, TM1 and TM2, exist in all CoVs. The three-dimensional structures of domains in the N-terminal two thirds of Nsp3 have been investigated by X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy since the outbreaks of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003 as well as Middle-East Respiratory Syndrome coronavirus (MERS-CoV) in 2012. In this review, the structures and functions of these domains of Nsp3 are discussed in depth., Highlights • Nonstructural protein 3 (∼200 kD) is a multifunctional protein comprising up to 16 different domains and regions. • Nsp3 binds to viral RNA, nucleocapsid protein, as well as other viral proteins, and participates in polyprotein processing. • The papain-like protease of Nsp3 is an established target for new antivirals. • Through its de-ADP-ribosylating, de-ubiquitinating, and de-ISGylating activities, Nsp3 counteracts host innate immunity. • Structural data are available for the N-terminal two thirds of Nsp3, but domains in the remainder are poorly characterized.

Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate and adaptive immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular deubiquitinating enzymes (DUBs), which remove ubiquitin from cellular targets and depolymerize polyubiquitin chains. The importance of protein ubiquitination to host immunity has been underscored by the discovery of viruses that encode proteases with deubiquitinating activity, many of which have been demonstrated to actively corrupt cellular ubiquitin-dependent processes to suppress innate antiviral responses and promote viral replication. DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. Here, we provide an overview of the structural biology of these fascinating viral enzymes and their role innate immune evasion and viral replication., Graphical Abstract Image 1, Highlights • Activation of host innate antiviral responses is largely ubiquitin-dependent. • Virus-encoded DUBs can modulate innate immune signaling. • Analysis of structurally diverse DUBs of DNA and RNA viruses. • Viral DUBs are critical for virus replication and pathogenesis. • Therapeutic strategies and vaccination approaches targeting viral DUBs.

Highlights • Nidoviral RNA signals and protein co-factors control RdRp processivity and fidelity. • The RdRp directs a complex process of replication and subgenomic mRNA synthesis. • A unique N-terminal nucleotidyl transferase is connected to the nidovirus RdRp. • Nidoviral RNA synthesis depends on recruiting host cell factors and infrastructure. • The nidovirus RdRp is an important yet poorly explored antiviral drug target., Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales. Nidoviruses are characterized by their polycistronic plus-stranded RNA genome, the production of subgenomic mRNAs and the conservation of a specific array of replicase domains, including key RNA-synthesizing enzymes. Coronaviruses (26–34 kilobases) have the largest known RNA genomes and their replication presumably requires a processive RNA-dependent RNA polymerase (RdRp) and enzymatic functions that suppress the consequences of the typically high error rate of viral RdRps. The arteriviruses have significantly smaller genomes and form an intriguing package with the coronaviruses to analyse viral RdRp evolution and function. The RdRp domain of nidoviruses resides in a cleavage product of the replicase polyprotein named non-structural protein (nsp) 12 in coronaviruses and nsp9 in arteriviruses. In all nidoviruses, the C-terminal RdRp domain is linked to a conserved N-terminal domain, which has been coined NiRAN (nidovirus RdRp-associated nucleotidyl transferase). Although no structural information is available, the functional characterization of the nidovirus RdRp and the larger enzyme complex of which it is part, has progressed significantly over the past decade. In coronaviruses several smaller, non-enzymatic nsps were characterized that direct RdRp function, while a 3′-to-5′ exoribonuclease activity in nsp14 was implicated in fidelity. In arteriviruses, the nsp1 subunit was found to maintain the balance between genome replication and subgenomic mRNA production. Understanding RdRp behaviour and interactions during RNA synthesis and subsequent processing will be key to rationalising the evolutionary success of nidoviruses and the development of antiviral strategies.

Coronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts., Graphical abstract Image 1, Highlights • The X-ray structure of MERS-CoV papain-like protease bound to full-length human ISG15 was determined. • This is the first structure of a viral-encoded Ubiquitin Specific Protease bound to full-length ISG15. • Structure-guided protein design was used to generate MERS PLpro mutants that have attenuated DUB and/or deISG activities. • Side chain properties, charge and/or steric bulk, are critical in designing mutants with desired substrate specificities. • These new MERS PLpro mutants will aid in delineating the importance of DUB versus deISG activity in virus-infected cells.

The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1]. USP3 is a histone H2A deubiquitinating enzyme (DUB) that regulates ubiquitin-dependent DDR in response to DNA double-strand breaks (Nicassio et al., 2007; Doil et al., 2008) [2,3]. Deletion of USP3 in mice increases the incidence of spontaneous tumors and affects hematopoiesis [4]. In particular, Usp3-knockout mice show progressive loss of B and T cells and decreased functional potential of hematopoietic stem cells (HSCs) during aging. USP3-deficient cells, including HSCs, display enhanced histone ubiquitination, accumulate spontaneous DNA damage and are hypersensitive to ionizing radiation (Lancini et al., 2014) [4]. To address whether USP3 loss leads to deregulation of specific molecular pathways relevant to HSC homeostasis and/or B cell development, we have employed the RNA-sequencing technology and investigated transcriptional differences between wild-type and Usp3Δ/Δ LSK, naïve B cells or in vitro activated B cells. The data relate to the research article “Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells” (Lancini et al., 2014) [4]. The RNA-sequencing and analysis data sets have been deposited in NCBI׳s Gene Expression Omnibus (Edgar et al., 2002) [5] and are accessible through GEO Series accession number GSE58495 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58495). With this article, we present validation of the RNA-seq data set through quantitative real-time PCR and comparative analysis. Keywords: B cell, Deubiquitinating enzymes (DUBs), DNA damage response (DDR), Hematopoietic stem and progenitor cells (HSPC), Hematopoietic stem cells (HSC), LSK, RNA-seq, Transcriptional profiling, Ubiquitin, Ubiquitin-specific protease 3 (USP3), Histone H2A, DNA double-strand breaks (DSBs)

WOS: 000367338700040, PubMed ID: 26381917, Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathics., (C) 2015 The Authors. Published by Elsevier B.V., European Cooperation in Science and Technology (COST Action)European Cooperation in Science and Technology (COST) [BM1203/EU-ROS]; Kidney Research UKKidney Research UK (KRUK) [PDF3/2014], Several authors of this review were supported by the European Cooperation in Science and Technology (COST Action BM1203/EU-ROS). HKID is funded by Kidney Research UK (PDF3/2014).

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes., Defective proteasome 19S regulatory particles (RPs) were identified in rpn11f–m1, a proteasomal mutant with mitochondrial phenotypes. The Rpn11 subunit initiates assembly of a five-subunit lid module competent to integrate into pre-assembled base-20S core particle (CP), with subsequent recruitment of remaining lid subunits.