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AtAIRP5 RING E3 ubiquitin ligase was recently identified as a positive regulator of the abscisic acid (ABA)-mediated drought stress response by stimulating the degradation of serine carboxypeptidase-like 1. Here, we identified GDSL-type esterase/lipase 22 (AtGELP22) and AtGELP23 as additional interacting partners of AtAIRP5. Yeast two-hybrid, pull-down, co-immunoprecipitation, and ubiquitination analyses verified that AtGELP22 and AtGELP23 are ubiquitinated target proteins of AtAIRP5. AtGELP22 and AtGELP23 were colocalized with AtAIRP5 to punctate-like structures in the cytosolic fraction, in which PYK10 and NAI2, two ER body marker proteins, are localized. T-DNA insertion atgelp22 and atgelp23 single knockout mutant plants showed phenotypes indistinguishable from those of wild-type plants under ABA treatment. In contrast, RNAi-mediated cosuppression of AtGELP22 and AtGELP23 resulted in hypersensitive ABA-mediated stomatal movements and higher tolerance to drought stress than that of the single mutant and wild-type plants. Taken together, our results suggest that the putative GDSL-type esterases/lipases AtGELP22 and AtGELP23 act as redundant negative regulators of the ABA-mediated drought stress response in Arabidopsis., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Ubiquitin-proteasome dysfunction contributes to obesity-related metabolic disorders, such as diabetes and fatty liver disease. However, the regulation of ubiquitin-proteasome activity by insulin remains to be elucidated. Here, we show that prolonged insulin stimulation activates proteasome function even though it reduces the ubiquitinated proteins in H4IIEC3 hepatocytes. Looking for a pathway by which insulin inhibits ubiquitination, we found that hepatic expression of ubiquitin-specific protease 14 (USP14) was upregulated in the liver of patients with insulin resistance. Indeed, the USP14-specific inhibitor IU1 canceled the insulin-mediated reduction of ubiquitinated proteins. Furthermore, insulin-induced endoplasmic reticulum (ER) stress, which was canceled by IU1, suggesting that USP14 activity is involved in insulin-induced ER stress. Co-stimulation with insulin and IU1 for 2 hours upregulated the nuclear translocation of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), upregulated the expression of the lipogenic gene, fatty acid synthase ( Fasn ), and repressed the gluconeogenic genes. In conclusion, insulin activates proteasome function even though it inhibits protein ubiquitination by activating USP14 in hepatocytes. USP14 activation by insulin inhibits mature SREBP-1c while upregulating ER stress and the expression of genes involved in gluconeogenesis. Further understanding mechanisms underlying the USP14 activation and its pleiotropic effects may lead to therapeutic development for obesity-associated metabolic disorders, such as diabetes and fatty liver disease. SIGNIFICANCE STATEMENT: This study shows that insulin stimulation inhibits ubiquitination by activating USP14, independent of its effect on proteasome activity in hepatocytes. USP14 also downregulates the nuclear translocation of the lipogenic transcription factor SREBP-1c and upregulates the expression of genes involved in gluconeogenesis. Since USP14 is upregulated in the liver of insulin-resistant patients, understanding mechanisms underlying the USP14 activation and its pleiotropic effects will help develop treatments for metabolic disorders such as diabetes and fatty liver., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Gastric cancer remains the most prevalent and highly lethal disease worldwide. MAP4K4, a member of Ste20, plays an important role in various pathologies, including cancer. However, its role in gastric cancer is not yet fully elucidated. Therefore, this study aims to determine the tumor-promoting role of MAP4K4 in gastric cancer and whether it can be used as a new and reliable biomarker to predict the prognosis of gastric cancer. For this purpose, we divide the samples into high- and low-expression groups according to the expression level of MAP4K4. The association of MAP4K4 expression with prognosis is assessed using the Kaplan-Meier survival analysis. Furthermore, immune infiltration analysis using ESTIMATE is conducted to evaluate the tumor immune scores of the samples., Results: The findings reveal a significantly higher expression of MAP4K4 in tumor samples than in adjacent samples. The high-expression group was significantly enriched in tumor-related pathways, such as the PI3K-Akt signaling pathway. In addition, immune infiltration analysis revealed a positive correlation between immune scores and MAP4K4 expression. We also observed that miRNAs, such as miR-192-3p (R = -0.317, p -value 3.111 × 10 -9 ), miR-33b-5p (R= -0.238, p -value 1.166 × 10 -5 ), and miR-582-3p (R = -0.214, p -value 8.430 × 10 -5 ), had potential negative regulatory effects on MAP4K4. Moreover, we identified several transcription factors, ubiquitinated proteins, and interacting proteins that might regulate MAP4K4. The relationship between MAP4K4 and DNA methylation was also identified. Finally, we verified the high expression of MAP4K4 and its effect on promoting cancer., Conclusion: MAP4K4 might be closely related to gastric cancer's progression, invasion, and metastasis. Its high expression negatively impacts the prognosis of gastric cancer patients. This suggests MAP4K4 as an important prognostic factor for gastric cancer and could be regarded as a new potential prognostic detection and therapeutic target.

Background: According to the Global Cancer Statistics in 2020, the incidence and mortality of colorectal cancer (CRC) rank third and second among all tumors. The disturbance of ubiquitination plays an important role in the initiation and development of CRC, but the ubiquitinome of CRC cells and the survival-relevant ubiquitination are poorly understood., Methods: The ubiquitinome of CRC patients (n = 6) was characterized using our own data sets of proteomic and ubiquitin-proteomic examinations. Then, the probable survival-relevant ubiquitination was searched based on the analyses of data sets from public databases., Results: For the ubiquitinomic examination, we identified 1690 quantifiable sites and 870 quantifiable proteins. We found that the highly-ubiquitinated proteins (n ≥ 10) were specifically involved in the biological processes such as G-protein coupling, glycoprotein coupling, and antigen presentation. Also, we depicted five motif sequences frequently recognized by ubiquitin. Subsequently, we revealed that the ubiquitination content of 1172 proteins were up-regulated and 1700 proteins were down-regulated in CRC cells versus normal adjacent cells. We demonstrated that the differentially ubiquitinated proteins were relevant to the pathways including metabolism, immune regulation, and telomere maintenance. Then, integrated with the proteomic datasets from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) (n = 98), we revealed that the increased ubiquitination of FOCAD at Lys583 and Lys587 was potentially associated with patient survival. Finally, we depicted the mutation map of FOCAD and elucidated its potential functions on RNA localization and translation in CRC., Conclusions: The findings of this study described the ubiquitinome of CRC cells and identified abnormal ubiquitination(s) potentially affecting the patient survival, thereby offering new probable opportunities for clinical treatment., (© 2022. The Author(s).)

Charcot-Marie-Tooth (CMT) disease is the most common inherited neurodegenerative disorder with selective degeneration of peripheral nerves. Despite advances in identifying CMT-causing genes, the underlying molecular mechanism, particularly of selective degeneration of peripheral neurons remains to be elucidated. Since peripheral neurons are sensitive to multiple stresses, we hypothesized that daily repeated stress might be an essential contributor to the selective degeneration of peripheral neurons induced by CMT-causing mutations. Here, we mainly focused on the biological effects of the dominant missense mutation (S135F) in the 27-kDa small heat-shock protein HSPB1 under repeated heat shock. HSPB1 S135F presented hyperactive binding to both α-tubulin and acetylated α-tubulin during repeated heat shock when compared with the wild type. The aberrant interactions with tubulin prevented microtubule-based transport of heat shock-induced misfolded proteins for the formation of perinuclear aggresomes. Furthermore, the transport of autophagosomes along microtubules was also blocked. These results indicate that the autophagy pathway was disrupted, leading to an accumulation of ubiquitinated protein aggregates and a significant decrease in cell adaptation to repeated stress. Our findings provide novel insights into the molecular mechanisms of HSPB1 S135F -induced selective degeneration of peripheral neurons and perspectives for targeting autophagy as a promising therapeutic strategy for CMT neuropathy.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motor neurons in the brain and spinal cord. Recent studies have shown that mutations in SQSTM1 are linked to ALS. It has also been demonstrated that a systemic loss of SQSTM1 exacerbates disease phenotypes in an ALS mouse model. However, it is still unclear whether and how SQSTM1 in the central nervous system (CNS) specifically regulates ALS-associated disease phenotypes. To address this issue, we generated CNS-specific Sqstm1 deficient SOD1 H46R transgenic mice, and conducted gross phenotype analyses as well as the immunohistochemical and biochemical examinations of spinal cord tissues using these mice. CNS-specific SQSTM1 deficiency accelerated the disease onset and shortened the lifespan of SOD1 H46R mice. The CNS-specific SQSTM1 ablation also resulted in increased number of ubiquitin-positive aggregates, while their size rather became much smaller. Remarkably, ubiquitin-positive aggregates, which were usually present in extracellular space and/or neuropil in SOD1 H46R mice, were preferentially localized to soma and neurites of spinal neurons in CNS-specific SQSTM1 deficient SOD1 H46R mice. Next, to further clarify the function of SQSTM1 in neurons, we investigated the contribution of SQSTM1 to the accumulation of polyubiquitinated proteins in relation to the ubiquitin proteasome system (UPS) and the autophagy-endolysosomal system (APELS) in primary cultured motor neurons (PMNs). Loss of SQSTM1 in PMNs resulted in decreased accumulation of insoluble polyubiquitinated proteins, which was induced by simultaneous treatment with proteasome and lysosome inhibitors, suggesting a pivotal role of SQSTM1 in the formation of insoluble protein aggregates. However, SQSTM1 silencing had a limited impact on the susceptibility to proteasome and/or lysosome inhibitor-induced apoptosis in PMNs. Taken together, neuronal SQSTM1, whose functions are associated with both the UPS and APELS, might primarily regulate the distribution and accumulation of misfolded protein aggregates in the CNS, thereby protecting neurons from degeneration in mice., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ubiquitination is a major post-translational modification (PTM) involved in almost all eukaryotic biological processes and plays an essential role in plant response to pathogen infection. However, to date, large-scale profiling of the changes in the ubiquitome in response to pathogens, especially viruses, in wheat has not been reported. This study aimed to identify the ubiquitinated proteins involved in Chinese wheat mosaic virus (CWMV) infection in wheat using a combination of affinity enrichment and high-resolution liquid chromatography-tandem mass spectroscopy. The potential biological functions of these ubiquitinated proteins were further analyzed using bioinformatics. A total of 2297 lysine ubiquitination sites in 1255 proteins were identified in wheat infected with CWMV, of which 350 lysine ubiquitination sites in 192 proteins were differentially expressed. These ubiquitinated proteins were related to metabolic processes, responses to stress and hormones, plant-pathogen interactions, and ribosome pathways, as assessed via Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Furthermore, we found that the ubiquitination of Ta14-3-3 and TaHSP90, which are essential components of the innate immune system, was significantly enhanced during CWMV infection, which suggested that ubiquitination modification plays a vital role in the regulatory network of the host response to CWMV infection. In summary, our study puts forward a novel strategy for further probing the molecular mechanisms of CWMV infection. Our findings will inform future research to find better, innovative, and effective solutions to deal with CWMV infection in wheat, which is the most crucial and widely used cereal grain crop.

Background: Ubiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown., Results: Here, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice (Oryza sativa L.) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1-OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity., Conclusions: The rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3-substrate interactome in plants., (© 2022. The Author(s).)

Previously it was shown that cisplatin causes muscle atrophy. Under this condition, cisplatin increased the expression of atorogenes, such as muscle ring finger 1 and atrogin-1 (also known as muscle atrophy F-box protein), in mouse skeletal muscle. It was reported recently that ubiquitin (Ub) and ubiquitinated protein levels in skeletal muscle were also up-regulated in cisplatin-induced muscle atrophy, and cisplatin-induced ubiquitinated proteins were degraded by the 26S proteasome pathway. Eicosapentaenoic acid (EPA) is effective against skeletal muscle atrophy in mice. However, it is unclear how EPA suppresses the Ub-proteasome pathway. In this study, the effect of EPA on cisplatin-induced muscle atrophy in mice was examined. Mice were intraperitoneally injected with cisplatin or vehicle control once daily for 4 d. EPA or its vehicle was orally administered 30 min before cisplatin administration. Cisplatin systemic administration induced decrease in muscle mass, myofiber diameter, and increase in Ub genes and ubiquitinated proteins in mouse skeletal muscle were recovered by co-treatment with EPA. However, weight loss and up-regulated atrogenes induced by cisplatin were not changed by co-treatment with EPA in skeletal muscle. In this study, EPA attenuated cisplatin-induced muscle atrophy via down-regulation of up-regulated Ub gene expression. Although further clinical studies are needed, EPA administration can be effective in the development of muscle atrophy in cisplatin-treated patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48., Competing Interests: Declaration of interests J.A.M. serves on the scientific advisory board of 908 Devices and receives sponsored research support from AstraZeneca and Vertex. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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

The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. In this process, which is governed by cargo receptors, ubiquitinated proteins are condensed into larger structures and subsequently become targets for the autophagy machinery. Here we employ in vitro reconstitution and cell biology to define the roles of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substrates. We show that p62 is the major driver of ubiquitin condensate formation. NBR1 promotes condensate formation by equipping the p62-NBR1 heterooligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62-ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates., (© 2021. The Author(s).)

Long non-coding RNAs (lncRNAs) are a class of RNA transcripts longer than 200 nucleotides and mostly cannot be translated into proteins. Next-generation transcriptome sequencing of various cell types has enabled the annotation of tens of thousands of lncRNAs in human genome. Varying levels of evidence supports the implications of lncRNAs in the onset and progression of cancers. Ubiquitin is an evolutionarily conserved protein and could post-translationally mark a number of proteins. The most important proteolytic role of ubiquitination is degradation of substrate proteins by the 26S proteasome. Compiling evidences demonstrated that lncRNAs are involved in the accurate execution of protein stability programs via the ubiquitin-proteasome system. In the current review, we systematically summarize the detailed mechanisms how lncRNAs modulate ubiquitination of target proteins, regulate cancerous signaling pathways and control tumorigenesis of gastrointestinal cancers. Although there are still considerable studies on unraveling the complicated interactions between lncRNAs and proteins, we believe that lncRNAs are promising but challenging molecules which may strongly facilitate precision cancer therapeutics in the future., (Copyright © 2021 Elsevier Inc. All rights reserved.)

TRIM17 is a member of the TRIM family, a large class of RING-containing E3 ubiquitin-ligases. It is expressed at low levels in adult tissues, except in testis and in some brain regions. However, it can be highly induced in stress conditions which makes it a putative stress sensor required for the triggering of key cellular responses. As most TRIM members, TRIM17 can act as an E3 ubiquitin-ligase and promote the degradation by the proteasome of substrates such as the antiapoptotic protein MCL1. Intriguingly, TRIM17 can also prevent the ubiquitination of other proteins and stabilize them, by binding to other TRIM proteins and inhibiting their E3 ubiquitin-ligase activity. This duality of action confers several pivotal roles to TRIM17 in crucial cellular processes such as apoptosis, autophagy or cell division, but also in pathological conditions as diverse as Parkinson's disease or cancer. Here, in addition to recent data that endorse this duality, we review what is currently known from public databases and the literature about TRIM17 gene regulation and expression, TRIM17 protein structure and interactions, as well as its involvement in cell physiology and human disorders.

We previously demonstrated that cisplatin administration in mice induces muscle atrophy and an increase in the expression of two muscle-specific ubiquitin E3 ligase genes, muscle ring finger protein 1 (MuRF1), and atrophy gene-1 (atrogin-1), in skeletal muscle. Ubiquitination serves as a degradation signal in both the ubiquitin-proteasome and selective autophagy pathways. In the present study, we investigated changes in the expression of ubiquitin and ubiquitinated proteins and their degradation pathways. Ubiquitin and ubiquitinated protein levels were increased by cisplatin compared with those in the vehicle and dietary restriction (DR) groups. To quantify the levels of ubiquitin and ubiquitinated proteins, we conducted a dot blot assay using an anti-ubiquitin antibody. The expression of ubiquitin was also significantly increased by cisplatin compared with that in the vehicle and DR groups. Since the ubiquitin proteins were upregulated by cisplatin, we measured the mRNA levels of the ubiquitin genes: Ubb, Ubc, Rps27a, and Uba52. All these four genes were increased by cisplatin administration compared with those in both the vehicle-treated and DR groups in quadriceps muscle tissue. The anti-ubiquitin antibody-sensitive bands increased when C2C12 myotubes were treated with cisplatin. Furthermore, MG-132 (26 s proteasome inhibitor), but not bafilomycin A1 (autophagy inhibitor), caused a further increase in expression. In conclusion, ubiquitin and ubiquitinated proteins are upregulated in cisplatin-induced muscle atrophy. Cisplatin-induced ubiquitinated proteins are degraded by the 26 s proteasome pathway., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of CLN4 by expressing CLN4 mutant human CSPα (hCSPα) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking., Competing Interests: EI, JP, SK, MT, YZ, SC, KZ No competing interests declared, (© 2019, Imler et al.)

Drought stress often affects the expression of genes and proteins in tea plants. However, the global profiling of ubiquitinated (Kub) proteins in tea plants remains unearthed. Here, we performed the ubiquitome in tea leaves under drought stress using antibody-based affinity enrichment coupled with LC-MS/MS analysis. In total, 1,409 lysine Kub sites in 781 proteins were identified, of which 14 sites in 12 proteins were up-regulated and 123 sites in 91 proteins down-regulated under drought stress. The identified Kub proteins were mainly located in the cytosol (31%), chloroplast (27%) and nuclear (19%). Moreover, 5 conserved motifs in EK ub , EXXXK ub , K ub D, K ub E and K ub A were extracted. Several Kub sites in ubiquitin-mediated proteolysis-related proteins, including RGLG2, UBC36, UEV1D, RPN10 and PSMC2, might affect protein degradation and DNA repair. Plenty of Kub proteins related to catechins biosynthesis, including PAL, CHS, CHI and F3H, were positively correlated with each other due to their co-expression and co-localization. Furthermore, some Kub proteins involved in carbohydrate and amino acid metabolism, including FBPase, FBA and GAD1, might promote sucrose, fructose and GABA accumulation in tea leaves under drought stress. Our study preliminarily revealed the global profiling of Kub proteins in metabolic pathways and provided an important resource for further study on the functions of Kub proteins in tea plants.

In higher eukaryotes, DNA damage repair response pathways are orchestrated by several molecular signals including ubiquitination. In particular the repair of DNA interstrand crosslinks, toxic to transcription and replication processes, involve the activation of the Fanconi anemia repair pathway. At the heart of this pathway lies the monoubiquitination of FANCD2 and FANCI proteins, which triggers the recruitment of DNA repair factors. A major road block in our understanding of this fundamental repair pathway arises from the challenge with generating sufficient quantities of site-specifically monoubiquitinated FANCD2 and FANCI proteins to enable mechanistic and molecular studies. Current in vitro methods rely on the purification of a large (~0.8MDa), multiprotein E3 complex that can only partially monoubiquitinate a FANCD2-FANCI-DNA complex. In this chapter, we describe detailed protocols for the preparation of homogeneously and natively monoubiquitinated FANCD2 and FANCI proteins in isolation. The method relies on the use of a minimal E3 module and an engineered E2 variant that together drive site-specific ubiquitination of the isolated substrates, without the requirement of DNA cofactors. Using the enzymatic approach, we also demonstrate how added functionalities such as a fluorescently labeled ubiquitin can be conjugated on the FANCD2 and FANCI substrates, thus enabling multiple downstream applications., (© 2019 Elsevier Inc. All rights reserved.)

Motivation: Posttranslational modification (PTM) is a biological mechanism involved in the enzymatic modification of proteins after translation by ribosomes. Two or more modifications occurring at one residue can be transformed into a multi-label system. Two or more simultaneous modifications on a residue is more common than single PTMs. Lysine residues in proteins can be subjected to a variety of PTMs, such as ubiquitination, acetylation, sumoylation, methylation, and succinylation. Identification of uncharacterized sequences in proteins is a highly significant and state-of-the-art issue. Notably, in order to provide a method of processing multi-label sequences of lysine residues, it is highly desirable to develop computational methods to predict lysine acetylation and sumoylation modifications., Results: In this paper, we first launched an integrated approach, known as the five-step prediction method (FSPM), to solve the problem effectively by (1) using one-sided selection (OSS) to deal with imbalanced data, (2) extracting binary features from protein sequences, (3) incorporating binary relevance, classifier chains and multi-class transformation methods to simplify multi-label problems, (4) constructing different classifiers, and (5) implementing cross-validation and evaluating these classifiers. In 10-fold cross-validation, FSPM achieved an accuracy of 61.49% and an absolute-true rate of 60.17%. The results showed that FSPM is accurate and could be used as a powerful engine in multi-label systems. We also conducted a variety of statistical analyses of the predicted results to discuss the biological functions of lysine acetylation and sumoylation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Misfolded protein aggregates are the hallmark of Amyotrophic Lateral Sclerosis (ALS) which suggests involvement of protein homeostasis pathways in etiology of ALS. However, status of protein homeostasis in peripheral blood of ALS is not well established. We analyzed expression levels of key genes of proteostasis pathways in peripheral blood mononuclear cells (PBMCs) of sporadic ALS (sALS) patients and healthy controls. Increased protein carbonylation was observed in patients reflecting oxidative damage in PBMCs. We observed increased transcript and protein levels of GRP78 suggesting Endoplasmic reticulum (ER) insult to cells. Further, significant upregulation of spliced XBP1 and two stress sensors: IRE1α/ERN1 and ATF6 indicated induction of unfolded protein response (UPR). Genes involved in autophagosome initiation (ULK1, ULK2, ATG13); nucleation and elongation (BECLIN1, ATG7, ATG16L1, ATG5, ATG10) and vesicular trafficking genes were significantly increased in patients. Increased lipidation of LC3 validated induction of autophagy. Accumulation of low molecular weight ubiquitinated proteins in patients suggested deregulation of proteasome (UPS) pathway. In addition, cytosolic chaperones (HSP70 and HSP27) and HSF1 were elevated in patients. Increased TDP43 indicated role of TDP43 in disease pathology. Our findings suggest that there is oxidative insult and upregulation of UPR, vesicular trafficking and autophagy in PBMCs of sALS patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5'- O -(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30-40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins ( e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Basal autophagy plays an essential role as a protein quality control system. Although it has been demonstrated that the loss of autophagy results in the accumulation of ubiquitin-positive aggregates and the development of neurodegenerative diseases, the precise autophagy substrate(s) remain unclear. Here, we determined whether ubiquitinated proteins are direct substrates for basal autophagy using a fluorescent ratiometric probe for ubiquitin. We show that the degradation of polyubiquitinated proteins is not dependent on basal autophagy. Although ubiquitin-positive aggregates are observed in autophagy knockout cultured cells, the aggregates consist of soluble and mobile polyubiquitinated proteins, which are trapped by p62 without an increase in the total amount of ubiquitinated proteins. These results suggest that ubiquitinated proteins are not major targets for basal autophagy., (© 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In mammalian cells, the 26S proteasomes vary in composition. In addition to the standard 28 subunits in the 20S core particle and 19 subunits in each 19S regulatory particle, a small fraction (about 10-20% in our preparations) also contains the deubiquitinating enzyme Usp14/Ubp6, which regulates proteasome activity, and the ubiquitin ligase, Ube3c/Hul5, which enhances proteasomal processivity. When degradation of ubiquitinated proteins in cells was inhibited, levels of Usp14 and Ube3c on proteasomes increased within minutes. Conversely, when protein ubiquitination was prevented, or when purified proteasomes hydrolyzed the associated ubiquitin conjugates, Usp14 and Ube3c dissociated rapidly (unlike other 26S subunits), but the inhibitor ubiquitin aldehyde slowed their dissociation. Recombinant Usp14 associated with purified proteasomes preferentially if they contained ubiquitin conjugates. In cells or extracts, adding Usp14 inhibitors (IU-1 or ubiquitin aldehyde) enhanced Usp14 and Ube3c binding further. Thus, in the substrate- or the inhibitor-bound conformations, Usp14 showed higher affinity for proteasomes and surprisingly enhanced Ube3c binding. Moreover, adding ubiquitinated proteins to cell extracts stimulated proteasome binding of both enzymes. Thus, Usp14 and Ube3c cycle together on and off proteasomes, and the presence of ubiquitinated substrates promotes their association. This mechanism enables proteasome activity to adapt to the supply of substrates., Competing Interests: The authors declare no conflict of interest.

It is still unclear whether there exist functional constraints on the evolution of protein ubiquitination sites, because most previous studies regarded all protein ubiquitination sites as a whole or only focused on limited structural properties. We tried to clarify the relation between functional constraints and ubiquitination sites evolution. We investigated the evolutionary conservation of human ubiquitination sites in a broad evolutionary scale from G. gorilla to S. pombe, and we found that in organisms originated after the divergence of vertebrate, ubiquitination sites are more conserved than their flanking regions, while the opposite tendency is observed before this divergence time. By grouping the ubiquitination proteins into different functional categories, we confirm that many functional constraints like certain molecular functions, protein tissue expression specificity and protein connectivity in protein-protein interaction network enhance the evolutionary conservation of ubiquitination sites. Furthermore, by analyzing the gains of ubiquitination sites at different divergence time and their functional characters, we validate that the emergences of ubiquitination sites at different evolutionary time were also affected by the uncovered functional constraints. The above results suggest that functional constraints on the adaptive evolution of ubiquitination sites increase the opportunity for ubiquitination to synthetically regulate various cellular and developmental processes during evolution.

The post-translational modification of proteins provides a rapid and versatile system for regulating all signalling pathways. Protein ubiquitination is one such type of post-translational modification involved in controlling numerous cellular processes. The unique ability of ubiquitin to form polyubiquitin chains creates a highly complex code responsible for different subsequent signalling outcomes. Specialised enzymes ('writers') generate the ubiquitin code, whereas other enzymes ('erasers') disassemble it. Importantly, the ubiquitin code is deciphered by different ubiquitin-binding proteins ('readers') functioning to elicit particular cellular responses. Ten years ago, the methionine1 (Met1)-linked (linear) polyubiquitin code was first identified and the intervening years have witnessed a seismic shift in our understanding of Met1-linked polyubiquitin in cellular processes, particularly inflammatory signalling. This review will discuss the molecular mechanisms of specificity determination within Met1-linked polyubiquitin signalling., (© 2016 The Author(s).)

Type I interferon (IFN-I) mediated innate immune response controls virus infections by inducing the expression of interferon stimulated genes (ISGs). Although ubiquitination plays key roles in immune signaling regulation, a human genome-wide understanding of the role of E3 ubiquitin ligases in interferon mediated ISG induction is lacking. Here, we report a genome-wide profiling of the effect of ectopic expression of 521 E3 ubiquitin ligases and substrate recognition subunits encoded in the human genome (which constitutes 84.4% of all ubiquitination related genes encoded in the human genome, hereafter termed Human Ubiquitome) on IFNβ mediated induction of interferon stimulated DNA response element (ISRE) driven reporter activity. We identified 96 and 42 genes of the human ubiquitome as novel negative and positive regulators of interferon signaling respectively. Furthermore, we characterized DCST1 as a novel E3 ubiquitin ligase negatively regulating interferon response. Ectopic expression and gene silencing of DCST1 respectively attenuated and increased ISRE reporter activity. DCST1 regulated Type I interferon signaling by interacting with and promoting ubiquitination-mediated degradation of STAT2, an essential component of antiviral gene induction. In summary, this study provided a systems level view on the role of human ubiquitination associated genes in Type I interferon response.

Acyl-CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is one of the two DGAT enzymes that catalyzes the synthesis of triacylglycerol, which is an important form of stored energy for eukaryotic organisms. There is currently limited information available regarding how DGAT2 and triacylglycerol synthesis are regulated. Recent studies have indicated that DGAT2 can be regulated by changes in gene expression. How DGAT2 is regulated post-transcriptionally remains less clear. In this study, we demonstrated that DGAT2 is a very unstable protein and is rapidly degraded in an ubiquitin-dependent manner via the proteasome. Many of the 25 lysines present in DGAT2 appeared to be involved in promoting its degradation. However, the six C-terminal lysines were the most important in regulating stability. We also demonstrated that acyl-CoA:monoacylglycerol acyltransferase (MGAT)-2, an enzyme with extensive sequence homology to DGAT2 that catalyzes the synthesis of diacylglycerol, was also ubiquitinated. However, MGAT2 was found to be much more stable than DGAT2. Interestingly, when co-expressed, MGAT2 appeared to stabilize DGAT2. Finally, we found that both DGAT2 and MGAT2 are substrates of the endoplasmic reticulum-associated degradation pathway., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Elevated valosin containing protein (VCP/p97) levels promote the progression of non-small cell lung carcinoma (NSCLC). Although many VCP inhibitors are available, most of these therapeutic compounds have low specificity for targeted tumor cell delivery. Hence, the primary aim of this study was to evaluate the in vitro efficacy of dendrimer-encapsulated potent VCP-inhibitor drug in controlling non-small cell lung carcinoma (NSCLC) progression. The VCP inhibitor(s) (either in their pure form or encapsulated in generation-4 PAMAM-dendrimer with hydroxyl surface) were tested for their in vitro efficacy in modulating H1299 (NSCLC cells) proliferation, migration, invasion, apoptosis and cell cycle progression. Our results show that VCP inhibition by DBeQ was significantly more potent than NMS-873 as evident by decreased cell proliferation (p<0.0001, MTT-assay) and migration (p<0.05; scratch-assay), and increased apoptosis (p<0.05; caspase-3/7-assay) as compared to untreated control cells. Next, we found that dendrimer-encapsulated DBeQ (DDNDBeQ) treatment increased ubiquitinated-protein accumulation in soluble protein-fraction (immunoblotting) of H1299 cells as compared to DDN-control, implying the effectiveness of DBeQ in proteostasis-inhibition. We verified by immunostaining that DDNDBeQ treatment increases accumulation of ubiquitinated-proteins that co-localizes with an ER-marker, KDEL. We observed that proteostasis-inhibition with DDNDBeQ, significantly decreased cell migration rate (scratch-assay and transwell-invasion) as compared to the control-DDN treatment (p<0.05). Moreover, DDNDBeQ treatment showed a significant decrease in cell proliferation (p<0.01, MTT-assay) and increased caspase-3/7 mediated apoptotic cell death (p<0.05) as compared to DDN-control. This was further verified by cell cycle analysis (propidium-iodide-staining) that demonstrated significant cell cycle arrest in the G2/M-phase (p<0.001) by DDNDBeQ treatment as compared to control-DDN. Moreover, we confirmed by clonogenic-assay that DDNDBeQ treatment significantly (p<0.001) inhibits H1299 colony-formation as compared to control/DDN. Overall, encapsulation of potent VCP-inhibitor DBeQ into a dendrimer allows selective VCP-mediated proteostasis-inhibition for controlling NSCLC-tumor growth and progression to allow tumor-targeted sustained drug delivery.

The endosomal sorting complexes required for transport (ESCRT) pathway facilitates multiple fundamental membrane remodeling events. Previously, we determined X-ray crystal structures of ESCRT-III subunit Snf7, the yeast CHMP4 ortholog, in its active and polymeric state (Tang et al., 2015). However, how ESCRT-III activation is coordinated by the upstream ESCRT components at endosomes remains unclear. Here, we provide a molecular explanation for the functional divergence of structurally similar ESCRT-III subunits. We characterize novel mutations in ESCRT-III Snf7 that trigger activation, and identify a novel role of Bro1, the yeast ALIX ortholog, in Snf7 assembly. We show that upstream ESCRTs regulate Snf7 activation at both its N-terminal core domain and the C-terminus α6 helix through two parallel ubiquitin-dependent pathways: the ESCRT-I-ESCRT-II-Vps20 pathway and the ESCRT-0-Bro1 pathway. We therefore provide an enhanced understanding for the activation of the spatially unique ESCRT-III-mediated membrane remodeling.

Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.

Introduction: The motor system is selectively vulnerable to mutations in the ubiquitously expressed aggregation-prone enzyme superoxide dismutase-1 (SOD1)., Results: Autophagy clears aggregates, and factors involved in the process were analyzed in multiple areas of the CNS from human control subjects (n = 10) and amyotrophic lateral sclerosis (ALS) patients (n = 18) with or without SOD1 mutations. In control subjects, the key regulatory protein Beclin 1 and downstream factors were remarkably scarce in spinal motor areas. In ALS patients, there was evidence of moderate autophagy activation and also dysregulation. These changes were largest in SOD1 mutation carriers. To explore consequences of low autophagy capacity, effects of a heterozygous deletion of Beclin 1 were examined in ALS mouse models expressing mutant SOD1s. This caused earlier SOD1 aggregation, onset of symptoms, motor neuron loss, and a markedly shortened survival. In contrast, the levels of soluble misfolded SOD1 species were reduced., Conclusions: The findings suggest that an inherent low autophagy capacity might cause the vulnerability of the motor system, and that SOD1 aggregation plays a crucial role in the pathogenesis.

Background: Our previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles: DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, we sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine., Methods: The ubiquitin binding protein Vx3(A7) was used to isolate ubiquitinated proteins from EL4 and B16-F10 tumor cells after blocking their proteasomal degradation pathway. C57BL/6 mice were vaccinated with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA. Anti-tumor efficacy of Ub-enriched proteins vaccine was evaluated by monitoring tumor growth in established tumor mice models. Graphpad Prism 5.0 was used for all statistical analysis., Results: We found that after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN-γ in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-γ level than that via subcutaneous injection. Moreover, the level of secreted IFN-γ in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-γ when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Furthermore, Ub-enriched proteins vaccine showed a significant inhibitory effect on in vivo growth of homologous tumor, as well as allogeneic tumor, compared with Ub-depleted proteins and tumor cell lysate. Tumor growth was regressed after three times of vaccination with Ub-enriched proteins in contrast to other groups., Conclusion: These results indicated that Ub-enriched proteins isolated from tumor cells may have a potential as a potent vaccine for immunotherapy against cancer.

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.

Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities we analyzed the function of Drosophila dRYBP, a conserved PcG- and trxG-associated protein. We show that dRYBP is itself ubiquitylated and binds ubiquitylated proteins. Additionally we show that dRYBP maintains H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further we show that dRYBP interacts with the repressive SCE and dKDM2 proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, our results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. Our findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development.

Background and Aims: Hyperhomocysteinemia (HHcy) is associated with cardiovascular diseases and is thought to induce endogenous oxidative stress and causes many cellular damages. Proteasome that degrades oxidized and ubiquitinated proteins can regulate the cellular response to oxidative stress. We aimed to investigate whether hyperhomocysteinemia induces oxidative stress and alters proteasome function and composition in heart and aorta tissues of rat., Methods and Results: To create hyperhomocysteinemia, male Wistar rats (Pasteur Institute-Algiers) were received daily intraperitoneal injections of dl-homocysteine (0.6-1.2μM/g body weight) for 3weeks. Biomarkers of oxidative stress (malondialdehyde (MDA), protein carbonyl (PC), superoxide dismutase (SOD) and catalase (CAT)) were first measured by biochemical methods and tissue damages by histological sections. Proteasome activities were quantitated using fluorogenic synthetic peptides; ubiquitinated proteins and proteasome subunits expression were then evaluated by SDS PAGE and Western blot analysis. We showed increased MDA and PC but decreased SOD and CAT levels both in plasma, heart and aorta accompanied by histological changes. A significant decrease of proteasome activities was observed in heart, whereas proteasome activity was not affected in aorta. However proteasome composition was altered in both tissues, as the accumulation of ubiquitinated proteins., Conclusion: Data demonstrated an alteration of the ubiquitin-proteasome system in hyperhomocysteinemia as a result of accumulating oxidized and ubiquitinated proteins in response to oxidative stress. Further studies must be conducted to better understanding mechanisms responsible of proteasome alterations in hyperhomocysteinemia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Methionine is a limiting essential amino acid in most plant-based ingredients of fish feed. In the present study, we aimed to determine the effect of dietary methionine concentrations on several main factors involved in the regulation of mRNA translation and the two major proteolytic pathways (ubiquitin-proteasome and autophagy-lysosomal) in the white muscle of rainbow trout (Oncorhynchus mykiss). The fish were fed for 6 weeks one of the three isonitrogenous diets providing three different methionine concentrations (deficient (DEF), adequate (ADQ) and excess (EXC)). At the end of the experiment, the fish fed the DEF diet had a significantly lower body weight and feed efficiency compared with those fed the EXC and ADQ diets. This reduction in the growth of fish fed the DEF diet was accompanied by a decrease in the activation of the translation initiation factors ribosomal protein S6 and eIF2α. The levels of the main autophagy-related markers (LC3-II and beclin 1) as well as the expression of several autophagy genes (atg4b, atg12 l, Uvrag, SQSTM1, Mul1 and Bnip3) were higher in the white muscle of fish fed the DEF diet. Similarly, the mRNA levels of several proteasome-related genes (Fbx32, MuRF2, MuRF3, ZNF216 and Trim32) were significantly up-regulated by methionine limitation. Together, these results extend our understanding of mechanisms regulating the reduction of muscle growth induced by dietary methionine deficiency, providing valuable information on the biomarkers of the effects of low-fishmeal diets.

Monoubiquitination of histone H2B has emerged as an important chromatin modification with roles not only in transcription but also in cell differentiation, DNA repair or mRNA processing. Recently, the genome-wide distribution of histone H2B ubiquitination in different organisms has been reported. In this review we discuss the mechanisms regulating H2B ubiquitination and its downstream effectors as well as the suggested functions for this mark in light of these recent studies.:, (© 2014 Elsevier Ltd . All rights reserved.)

In the past decade, ubiquitination has been well documented to have multifaceted roles in regulating NF-κB activation in mammals. However, its function, especially how deubiquitinating enzymes balance the NF-κB activation, remains largely elusive in invertebrates. Investigating bbtA20 and its binding proteins, bbt A20-binding inhibitor of NF-κB (bbtABIN1) and bbtABIN2, in Chinese amphioxus Branchiostoma belcheri tsingtauense, we found that bbtABIN2 can colocalize and compete with bbt TNF receptor-associated factor 6 to connect the K63-linked polyubiquitin chains, whereas bbtABIN1 physically links bbtA20 to bbt NF-κB essential modulator (bbtNEMO) to facilitate the K48-linked ubiquitination of bbtNEMO. Similar to human A20, bbtA20 is a dual enzyme that removes the K63-linked polyubiquitin chains and builds the K48-linked polyubiquitin chains on bbt receptor-interacting serine/threonine protein kinase 1b, leading to the inhibition of NF-κB signaling. Our study not only suggests that ubiquitination is an ancient strategy in regulating NF-κB activation but also provides the first evidence, to our knowledge, for ABINs/A20-mediated inhibition of NF-κB via modifying the ubiquitinated proteins in a basal chordate, adding information on the stepwise development of vertebrate innate immune signaling.

In the present study we have used a transgenic mice overexpressing an amyloidogenic protein, gamma-synuclein, in the nervous system to address the effect of dimebon on proteinopathy progression. Neuroprotective effect of chronic dimebon administration in these mice at organismal level was confirmed by the increased lifespan. Using histological and biochemical approaches we have demonstrated that dimebon reduced the number of amyloid inclusions in spinal cord of transgenic animals and decreased the content of ubiquitinated proteins in detergent-insoluble fractions. These effects are likely to occur at the level of aggregated protein species, since transgene expression was not altered. Thus, pathological protein aggregation serves as one of dimebon targets in neurodegeneration.

Heat shock proteins HSPA4L and HSPA4 are closely related members of the HSP110 family and act as cochaperones. We generated Hspa4l(-/-)Hspa4(-/-) mice to investigate a functional complementarity between HSPA4L and HSPA4 during embryonic development. Hspa4l(-/-)Hspa4(-/-) embryos exhibited marked pulmonary hypoplasia and neonatal death. Compared with lungs of wild-type, Hspa4l(-/-), and Hspa4(-/-) embryos, Hspa4l(-/-)Hspa4(-/-) lungs were characterized by diminished saccular spaces and increased mesenchymal septa. Mesenchymal hypercellularity was determined to be due to an increased cell proliferation index and decreased cell death. A significant increase in expression levels of prosurvival protein B cell leukemia/lymphoma 2 may be the cause for inhibition of apoptotic process in lungs of Hspa4(-/-)Hspa4l(-/-) embryos. Accumulation of glycogen and diminished expression of surfactant protein B, prosurfactant protein C, and aquaporin 5 in saccular epithelium suggested impaired maturation of type II and type I pneumocytes in the Hspa4l(-/-)Hspa4(-/-) lungs. Further experiments showed a significant accumulation of ubiquitinated proteins in the lungs of Hspa4l(-/-)Hspa4(-/-) embryos, indicating an impaired chaperone activity. Our study demonstrates that HSPA4L and HSPA4 collaborate in embryonic lung maturation, which is necessary for adaptation to air breathing at birth.

The Atg2-Atg18 complex acts in parallel to Atg8 and regulates Atg9 recycling from phagophore assembly site (PAS) during autophagy in yeast. Here we show that in Drosophila, both Atg9 and Atg18 are required for Atg8a puncta formation, unlike Atg2. Selective autophagic degradation of ubiquitinated proteins is mediated by Ref(2)P/p62. The transmembrane protein Atg9 accumulates on refractory to Sigma P (Ref(2)P) aggregates in Atg7, Atg8a and Atg2 mutants. No accumulation of Atg9 is seen on Ref(2)P in cells lacking Atg18 or Vps34 lipid kinase function, while the Atg1 complex subunit FIP200 is recruited. The simultaneous interaction of Atg18 with both Atg9 and Ref(2)P raises the possibility that Atg18 may facilitate selective degradation of ubiquitinated protein aggregates by autophagy., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Ubiquitin-specific proteases (USPs) consist of a family of deubiquitinating enzymes with more than 50 members in humans. Three of them, including USP37, contain ubiquitin-interacting motifs (UIMs), an ∼20-amino acid α-helical stretch that binds to ubiquitin. However, the roles of the UIMs in these USP enzymes remain unknown. USP37 has three UIMs, designated here as UIMs 1, 2, and 3 from the N-terminal side, between the Cys and His boxes comprising the catalytic core. Here, we examined the role of the UIMs in USP37 using its mutants that harbor mutations in the UIMs. The nuclear localization of USP37 was not affected by the UIM mutations. However, mutations in UIM2 or UIM3, but not UIM1, resulted in a significant decrease in USP37 binding to ubiquitinated proteins in the cell. In vitro, a region of USP37 harboring the three UIMs also bound to both Lys(48)-linked and Lys(63)-linked ubiquitin chains in a UIM2- and UIM3-dependent manner. The level of USP37 ubiquitination was also reduced by mutations in UIM2 or UIM3, suggesting their role in ubiquitination of USP37 itself. Finally, mutants lacking functional UIM2 or UIM3 exhibited a reduced isopeptidase activity toward ubiquitinated proteins in the cell and both Lys(48)-linked and Lys(63)-linked ubiquitin chains. These results suggested that the UIMs in USP37 contribute to the full enzymatic activity, but not ubiquitin chain substrate specificity, of USP37 possibly by holding the ubiquitin chain substrate in the proximity of the catalytic core.

We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin-associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using ligase traps of eight different F box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously unreported, substrates. Polyubiquitinated forms of candidate substrates associated with their cognate F box partner, but not other ligase traps. Interestingly, the four most abundant candidate substrates identified for the F box protein Saf1 were all vacuolar/lysosomal proteins. Analysis of one of these substrates, Prb1, showed that Saf1 selectively promotes ubiquitination of the unprocessed form of the zymogen. This suggests that Saf1 is part of a pathway that targets protein precursors for proteasomal degradation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

CD8(+) T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8(+) T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.

The quantitation of lysine post-translational modifications (PTMs) by bottom-up mass spectrometry is convoluted by the need for analogous derivatives and the production of different tryptic peptides from the unmodified and modified versions of a protein. Chemical derivatization of lysines prior to enzymatic digestion circumvents these problems and has proven to be a successful method for lysine PTM quantitation. The most notable example is the use of deuteroacetylation to quantitate lysine acetylation. In this work, levels of lysine ubiquitination were quantitated using a structurally homologous label that is chemically similar to the diglycine (GlyGly) tag, which is left at the ubiquitination site upon trypsinolysis. The LC-MS analysis of a chemically equivalent monoglycine (Gly) tag that is analogous to the corresponding GlyGly tag proved that the monoglycine tag can be used for the quantitation of ubiquitination. A glycinylation protocol was then established for the derivatization of proteins to label unmodified lysine residues with a single glycine tag. Ubiquitin multimers were used to show that after glycinylation and tryptic digestion, the mass spectrometric response from the corresponding analogous tagged peptides could be compared for relative quantitation. For a proof of principle regarding the applicability of this technique to the analysis of ubiquitination in biological samples, the glycinylation technique was used to quantitate the increase in monoubiquitinated histone H2B that is observed in yeast which lacks the enzyme responsible for deubiquitinating H2B-K123, compared to wild-type yeast.

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana associated molecule with the SH3 domain of STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with endosomal complex required for transport-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit vacuolar protein sorting2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.

The posttranslational addition of ubiquitin (Ub) profoundly controls the half-life, interactions, and/or trafficking of numerous intracellular proteins. Using stringent two-step affinity methods to purify Ub-protein conjugates followed by high-sensitivity mass spectrometry, we identified almost 950 ubiquitylation substrates in whole Arabidopsis thaliana seedlings. The list includes key factors regulating a wide range of biological processes, including metabolism, cellular transport, signal transduction, transcription, RNA biology, translation, and proteolysis. The ubiquitylation state of more than half of the targets increased after treating seedlings with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-Leu-al), strongly suggesting that Ub addition commits many to degradation by the 26S proteasome. Ub-attachment sites were resolved for a number of targets, including six of the seven Lys residues on Ub itself with a Lys-48>Lys-63>Lys-11>>>Lys-33/Lys-29/Lys-6 preference. However, little sequence consensus was detected among conjugation sites, indicating that the local environment has little influence on global ubiquitylation. Intriguingly, the level of Lys-11-linked Ub polymers increased substantially upon MG132 treatment, revealing that they might be important signals for proteasomal breakdown. Taken together, this proteomic analysis illustrates the breadth of plant processes affected by ubiquitylation and provides a deep data set of individual targets from which to explore the roles of Ub in various physiological and developmental pathways.