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Acupuncture has obvious therapeutic effect on intracerebral hemorrhage (ICH). miR-34a-5p regulated by acupuncture was found to attenuate neurological deficits in ICH. However, the underlying mechanisms are unclear. Ubiquitin-like 4A (UBL4A) has not been studied in ICH. SD rats were injected with autologous blood to induce ICH and treated with Baihui-penetrating-Qubin acupuncture. Acupuncture resulted in an increase in forelimb placing test scores, and a decrease in corner test scores and brain water content of ICH rats. Histopathological examination showed that acupuncture inhibited ICH-induced inflammation, decreased damaged neurons and increased UBL4A expression. UBL4A overexpression increased cell viability, inhibited apoptosis, reduced reactive oxygen species (ROS) level and increased manganese superoxide dismutase (MnSOD) activity, mitochondrial membrane potential and mtDNA level in rat embryonic primary cortical neurons. miR-34a-5p knockdown increased UBL4A expression, apoptosis rate and ROS level in hemin-treated neurons. Dual luciferase assays showed that miR-34a-5p bound to UBL4A. Apoptotic cells and ROS level were increased in hemin-treated neurons with UBL4A and miR-34a-5p knockdown. We firstly demonstrate the inhibitory effect of UBL4A on neuronal apoptosis, and the regulation relationship between UBL4A and miR-34a-5p. This study provides a new candidate target for ICH treatment and more basis for elucidating the molecular mechanism of acupuncture. In the future, we will conduct a deeper exploration of the effects of UBL4A on ICH.

SHANK-associated RH domain interacting protein (SHARPIN) plays an important role in carcinogenesis, as well as inflammation and immunity. Our study explored the effects and underlying mechanisms of SHARPIN in clear cell renal cell carcinoma (ccRCC). By analyzing The Cancer Genome Atlas database, we found that upregulated SHARPIN in patients with ccRCC led to a poor prognosis. Semiquantitative immunohistochemical analysis of clinical samples was carried out and the results suggested the positive association between SHARPIN and hypoxia-induced factor-2α (HIF-2α). Von Hippel-Lindau protein (pVHL) is a tumor suppressor that contributes to degrading HIF-2α. Mechanically, SHARPIN promoted the ubiquitination and proteasomal degradation of pVHL, resulting in the sustained activation of HIF-2α. The α and β domains of pVHL and ubiquitin-like domain of SHARPIN are required for the interaction. The knockdown of SHARPIN effectively inhibited acquired sorafenib resistance in ccRCC cell lines and tumor growth in xenograft models. In conclusion, our work reveals a novel posttranslational regulation of SHARPIN on pVHL, indicating that SHARPIN could be a potential target for ccRCC treatment., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Purpose: To investigate the potential function of FAT10 in the development of osteosarcoma (OS) and its mechanism., Methods: Relative level of FAT10 in OS specimens and cell lines was detected by qRT-PCR. The correlation between FAT10 level and clinical features of OS patients was assessed by χ2 test. After intervention of FAT10 in MG-63 and U2OS cells, changes of FAT10 level, cell viability, clonality and proliferative capacity were respectively detected by qRT-PCR, CCK-8, colony formation and EdU assay. Moreover, dynamic change of FAT10 in OS cells induced with pro-inflammatory factors was examined by qRT-PCR. Protein levels of FAT10, p-STAT1, p-STAT3 and p-STAT5 in OS cells induced with TNF-α were determined by Western blot. The JAK2 inhibitor AZ960 was used to further confirm the role of the JAK signaling in FAT10-regulated development of OS., Results: FAT10 was upregulated in OS specimens and cell lines, which was correlated to tumor size, WHO grade and distant metastasis of OS patients. Knockdown of FAT10 inhibited viability, clonality and proliferative capacity of MG-63 and U2OS cells. FAT10 was time-dependently upregulated in OS cells stimulated with IFN-γ and TNF-α, which was dose-dependently downregulated by the treatment of AZ960. Protein levels of FAT10, p-STAT1, p-STAT3 and p-STAT5 in OS cells induced with AZ960 were remarkably downregulated., Conclusion: FAT10 is upregulated in OS samples, which stimulates the development of OS by activating the JAK/STAT signaling pathway.

Interferon-stimulated gene 15 (ISG15) is a critical ubiquitin-like protein that can be conjugated to proteins via the ISGylation system to modify them posttranslationally. Furthermore, ISG15 can be detected as non-conjugated or free, intracellularly and/or extracellularly. Both conjugated and free ISG15 participate in different cancer types, including breast cancer. Here, we highlighted the findings on ISG15 and protein ISGylation, and their implications in the field of breast cancer research. ISG15 emerges as a central element in mammary tumors and may become a crucial protein in the strategies for detection, prognosis, and therapy of breast cancer.

Defective immune cell-mediated clearance of amyloid-beta (Aβ) and Aβ-associated inflammatory activation of immune cells are key contributors in pathogenesis of Alzheimer's disease (AD). However, the underlying mechanisms remain elusive. Shank-associated RH domain-interacting protein (SHARPIN) is a critical regulator of inflammatory response. Using in vitro cultures of THP-1-derived macrophages exposed to Aβ and AD patient-derived macrophages, we demonstrate the role of SHARPIN as an obligate regulator of Aβ phagocytosis and inflammation in macrophages. Specifically, Aβ-stimulated SHARPIN in THP-1 macrophages promoted Aβ phagocytosis and expression of proinflammatory markers. In addition, Aβ-stimulated SHARPIN in macrophages promoted neuronal cell-death in differentiated SHSY5Y neurons. Furthermore, we report a novel regulatory link between SHARPIN and the NLRP3 inflammasome in response to Aβ in THP-1 macrophages. In line with our in vitro observations, a strong positive association was demonstrated between levels of Aβ 42 in blood plasma of mild cognitive impairment and AD patients with SHARPIN expression in macrophages obtained from respective patient-derived peripheral blood mononuclear cells. Together, our findings show SHARPIN as a critical determinant in mediating macrophage response to Aβ and pathogenesis of AD., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death and is a suitable therapeutic target for B-cell lymphomas., (© 2020 by The American Society of Hematology.)

Purpose: IFN-stimulated gene (ISG) 15 is a type 1 IFN-induced protein and known to modify target proteins in a manner similar to ubiquitylation (protein conjugation by ISG15 is termed ISGylation). We sought to determine the role of ISG15 and its underlying mechanisms in corneal innate immune defense against Pseudomonas aeruginosa keratitis., Methods: ISG15 expression in cultured human corneal epithelial cells (HCECs) and mouse corneas was determined by PCR and Western blot analysis. Gene knockout mice were used to define the role of ISG15 signaling in controlling the severity of P. aeruginosa keratitis, which was assessed with photographing, clinical scoring, bacterial counting, myeloperoxidase assay, and quantitative PCR determination of cytokine expression. Integrin LFA-1 inhibitor was used to assess its involvement of ISG15 signaling in P. aeruginosa-infected corneas., Results: Heat-killed P. aeruginosa induced ISG15 expression in cultured HCECs and accumulation in the conditioned media. Isg15 deficiency accelerated keratitis progress, suppressed IFNγ and CXCL10, and promoted IL-1β while exhibiting no effects on IFNα expression. Moreover, exogenous ISG15 protected the corneas of wild-type mice from P. aeruginosa infection while markedly reducing the severity of P. aeruginosa keratitis in type 1 IFN-receptor knockout mice. Exogenous ISG15 increased bacteriostatic activity of B6 mouse corneal homogenates, and inhibition of LFA-1 exacerbated the severity of and abolished protective effects of ISG15 on P. aeruginosa keratitis., Conclusions: Type 1 INF-induced ISG15 regulates the innate immune response and greatly reduces the susceptibility of B6 mouse corneas to P. aeruginosa infection in an LFA-1-dependent manner.

Posttranslational protein modifications are known to be extensively involved in cancer, and a growing number of studies have revealed that the ubiquitin-like modifier FAT10 is directly involved in cancer development. FAT10 was found to be highly upregulated in various cancer types, such as glioma, hepatocellular carcinoma, breast cancer and gastrointestinal cancer. Protein FAT10ylation and interactions with FAT10 lead to the functional change of proteins, including proteasomal degradation, subcellular delocalization and stabilization, eventually having significant effects on cancer cell proliferation, invasion, metastasis and even tumorigenesis. In this review, we summarized the current knowledge on FAT10 and discussed its biological functions in cancer, as well as potential therapeutic strategies based on the FAT10 pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

SIZ1 (a SIZ/PIAS-type SUMO E3 ligase)-mediated small ubiquitin-like modifier (SUMO) modification of target proteins is important for various biological processes related to abiotic stress resistance in plants; however, little is known about its role in resistance toward iron (Fe) deficiency. Here, the SUMO E3 ligase MdSIZ1 was shown to be involved in the plasma membrane (PM) H + -ATPase-mediated response to Fe deficiency. Subsequently, a basic helix-loop-helix transcription factor, MdbHLH104 (a homolog of Arabidopsis bHLH104 in apple), which acts as a key component in regulating PM H + -ATPase-mediated rhizosphere acidification and Fe uptake in apples ( Malus domestica ), was identified as a direct target of MdSIZ1. MdSIZ1 directly sumoylated MdbHLH104 both in vitro and in vivo, especially under conditions of Fe deficiency, and this sumoylation was required for MdbHLH104 protein stability. Double substitution of K139R and K153R in MdbHLH104 blocked MdSIZ1-mediated sumoylation in vitro and in vivo, indicating that the K139 and K153 residues were the principal sites of SUMO conjugation. Moreover, the transcript level of the MdSIZ1 gene was substantially induced following Fe deficiency. MdSIZ1 overexpression exerted a positive influence on PM H + -ATPase-mediated rhizosphere acidification and Fe uptake. Our findings reveal an important role for sumoylation in the regulation of PM H + -ATPase-mediated rhizosphere acidification and Fe uptake during Fe deficiency in plants., (© 2019 American Society of Plant Biologists. All rights reserved.)

Attachment of the small ubiquitin-like modifier (SUMO) to substrate proteins modulates their turnover, activity, or interaction partners. However, how this SUMO conjugation activity concentrates the proteins involved and the substrates into uncharacterized nuclear bodies (NBs) remains poorly understood. Here, we characterized the requirements for SUMO NB formation and for their subsequent colocalization with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a master regulator of plant growth. COP1 activity results in degradation of transcription factors, which primes the transcriptional response that underlies elongation growth induced by darkness and high ambient temperatures (skoto- and thermomorphogenesis, respectively). SUMO conjugation activity alone was sufficient to target the SUMO machinery into NBs. Colocalization of these bodies with COP1 required, in addition to SUMO conjugation activity, a SUMO acceptor site in COP1 and the SUMO E3 ligase SAP and Miz 1 (SIZ1). We found that SIZ1 docks in the substrate-binding pocket of COP1 via two valine-proline peptide motifs, which represent a known interaction motif of COP1 substrates. The data reveal that SIZ1 physically connects COP1 and SUMO conjugation activity in the same NBs that can also contain the blue-light receptors CRYPTOCHROME 1 and CRYPTOCHROME 2. Our findings thus suggest that sumoylation stimulates COP1 activity within NBs. Moreover, the presence of SIZ1 and SUMO in these NBs explains how both the timing and amplitude of the high-temperature growth response is controlled. The strong colocalization of COP1 and SUMO in these NBs might also explain why many COP1 substrates are sumoylated., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

In the middle of the 20th century, it was postulated that degradation of intracellular proteins is a stochastic process. More than fifty years of intense studies have finally proven that protein degradation is a very complex and tightly regulated in time and space process that plays an incredibly important role in the vast majority of metabolic pathways. Degradation of more than a half of intracellular proteins is controlled by a hierarchically aligned and evolutionarily perfect system consisting of many components, the main ones being ubiquitin ligases and proteasomes, together referred to as the ubiquitin-proteasome system (UPS). The UPS includes more than 1000 individual components, and most of them are critical for the cell functioning and survival. In addition to the well-known signaling functions of ubiquitination, such as modification of substrates for proteasomal degradation and DNA repair, polyubiquitin (polyUb) chains are involved in other important cellular processes, e.g., cell cycle regulation, immunity, protein degradation in mitochondria, and even mRNA stability. This incredible variety of ubiquitination functions is related to the ubiquitin ability to form branching chains through the ε-amino group of any of seven lysine residues in its sequence. Deubiquitination is accomplished by proteins of the deubiquitinating enzyme family. The second main component of the UPS is proteasome, a multisubunit proteinase complex that, in addition to the degradation of functionally exhausted and damaged proteins, regulates many important cellular processes through controlled degradation of substrates, for example, transcription factors and cyclins. In addition to the ubiquitin-dependent-mediated degradation, there is also ubiquitin-independent degradation, when the proteolytic signal is either an intrinsic protein sequence or shuttle molecule. Protein hydrolysis is a critically important cellular function; therefore, any abnormalities in this process lead to systemic impairments further transforming into serious diseases, such as diabetes, malignant transformation, and neurodegenerative disorders (multiple sclerosis, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease and Huntington's disease). In this review, we discuss the mechanisms that orchestrate all components of the UPS, as well as the plurality of the fine-tuning pathways of proteasomal degradation.

Small ubiquitin-like modifier (SUMO) fusion technology is widely used in the production of heterologous proteins from prokaryotic system to aid in protein solubilization and refolding. Due to an extensive clinical application of human bone morphogenetic protein 2 (hBMP2) in bone augmentation, total RNA was isolated from human gingival tissue and mature gene was amplified through RT-PCR, cloned (pET21a), sequence analyzed, and submitted to GenBank (Accession no. KF250425). To obtain soluble expression, SUMO3 was tagged at the N-terminus of hBMP2 gene (pET21a/SUMO3-hBMP2), transferred in BL21 codon+, and ~ 40% soluble expression was obtained on induction with IPTG. The dimerized hBMP2 was confirmed with Western blot, native PAGE analysis, and purified by fast protein liquid chromatography with 0.5 M NaCl elution. The cleavage of SUMO3 tag from hBMP2 converted it to an insoluble form. Computational 3D structural analysis of the SUMO3-hBMP2 was performed and optimized by molecular dynamic simulation. Protein-protein interaction of SUMO3-hBMP2 with BMP2 receptor was carried out using HADDOCK and inferred stable interaction. The alkaline phosphatase assay of SUMO3-hBMP2 on C2C12 cells showed maximum 200-ng/ml dose-dependent activity. We conclude that SUMO3-tagged hBMP2 is more suited for generation of soluble form of the protein and addition of SUMO3 tag does not affect the functional activity of hBMP2.

Background: Ubiquitination is a major post-translational protein modification that regulates essentially all cellular and physiological pathways in eukaryotes. The ubiquitination process typically involves three distinct classes of enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). To date, a comprehensive identification and analysis of core components comprising of the whole soybean (Glycine max) ubiquitin system (UBS) has not been reported., Results: We performed a systematic, genome-wide analysis of genes that encode core members of the soybean UBS in this study. A total of 1431 genes were identified with high confidence to encode putative soybean UBS components, including 4 genes encoding E1s, 71 genes that encode the E2s, and 1356 genes encoding the E3-related components. Among the E3-encoding genes, 760 encode RING-type E3s, 124 encode U-box domain-containing E3s, and 472 encode F-box proteins. To find out whether the identified soybean UBS genes encode active enzymes, a set of genes were randomly selected and the enzymatic activities of their recombinant proteins were tested. Thioester assays indicated proteins encoded by the soybean E1 gene GmUBA1 and the majority of selected E2 genes are active E1 or E2 enzymes, respectively. Meanwhile, most of the purified RING and U-box domain-containing proteins displayed E3 activity in the in vitro ubiquitination assay. In addition, 1034 of the identified soybean UBS genes were found to express in at least one of 14 soybean tissues examined and the transcript level of 338 soybean USB genes were significantly changed after abiotic or biotic (Fusarium oxysporum and Rhizobium strains) stress treatment. Finally, the expression level of a large number of the identified soybean UBS-related genes was found significantly altered after soybean cyst nematode (SCN) treatment, suggesting the soybean UBS potentially plays an important role in soybean immunity against SCN., Conclusions: Our findings indicate the presence of a large and diverse number of core UBS proteins in the soybean genome, which suggests that target-specific modification by ubiquitin is a complex and important part of cellular and physiological regulation in soybean. We also revealed certain members of the soybean UBS may be involved in immunity against soybean cyst nematode (SCN). This study sets up an essential foundation for further functional characterization of the soybean UBS in various physiological processes, such as host immunity against SCN.

The TNF and IFN-γ-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) is most prominently expressed in immunological tissues but information regarding basal expression and inducibility of FAT10 in the different types of immune cells is still lacking. Hence, we investigated FAT10 mRNA expression in the major human and murine immune cell subsets, and FAT10 protein expression in human leukocytes. We isolated the different human leukocytes from peripheral blood and the murine immune cell subsets from spleen. The purified leukocytes were left untreated or stimulated with TNF and INF-γ or LPS to induce FAT10 followed by quantitative real-time PCR or western blot analysis. Basal expression of FAT10 mRNA and protein was generally low but strongly up-regulated by IFN-γ and TNF in all immune cell subsets. LPS treatment induced FAT10 expression marginally in human CD8 + T cells and murine granulocytes, but it increased Fat10 expression significantly in murine regulatory T cells. Yet, in human CD8 + T cells, natural killer cells, natural killer T cells, and dendritic cells, the FAT10 mRNA was expressed without induction. Similarly, murine macrophages, monocytes, and regulatory T cells expressed Fat10 in the absence of stimulation. In summary, our findings suggest particular functions of FAT10 in these cell types. Furthermore, we observed not only a cell type-specific but also a species-specific basal FAT10 expression profile. Our data will serve as a guideline for future investigations to further elucidate FAT10's role in the immune system.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors in China with a poor prognosis. Most ESCC patients were diagnosed at advanced stages, losing the opportunity for surgical excision. Hence, it remains a pressing work to identify biomarkers for early detection, prognosis prediction and targeting therapies in ESCC. Interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, and is involved in the post-translational modification (PTMs) of multiple proteins. However, the molecular functions of ISG15 in ESCC remain unclear. In this work, we found that ISG15 was aberrantly expressed in ESCC tissues and cell lines. Enhanced protein level of ISG15 promoted cellular malignant phenotypes including proliferation, migration, invasion and tumor formation in vivo. Consistently, reduction of ISG15 attenuated the cellular malignant phenotype in ESCC cell lines. Furthermore, gene-expression profiles suggested that the differentially expressed ISG15 affected the expression of a panel of genes enriched in the cell adherens junction, such as c-MET. Notably, as a secreted protein, the concentration of ISG15 was elevated in ESCC plasma than healthy individuals, acting as a potential diagnostic marker. Taken together, our results suggested a tumor promotion role of ISG15 in ESCC via c-MET/Fyn/β-catenin pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The intestinal epithelium constitutes a crucial defense to the potentially life-threatening effects of gut microbiota. However, due to a complex underlying vasculature, hypoperfusion and resultant tissue ischemia pose a particular risk to function and integrity of the epithelium. The small ubiquitin-like modifier (SUMO) conjugation pathway critically regulates adaptive responses to metabolic stress and is of particular significance in the gut, as inducible knockout of the SUMO-conjugating enzyme Ubc9 results in rapid intestinal epithelial disintegration. Here we analyzed the pattern of individual SUMO isoforms in intestinal epithelium and investigated their roles in intestinal ischemia/reperfusion (I/R) damage. Immunostaining revealed that epithelial SUMO2/3 expression was almost exclusively limited to crypt epithelial nuclei in unchallenged mice. However, intestinal I/R or overexpression of Ubc9 caused a remarkable enhancement of epithelial SUMO2/3 staining along the crypt-villus axis. Unexpectedly, a similar pattern was found in SUMO1 knockout mice. Ubc9 transgenic mice, but also SUMO1 knockout mice were protected from I/R injury as evidenced by better preserved barrier function and blunted inflammatory responses. PCR array analysis of microdissected villus-tip epithelia revealed a specific epithelial contribution to reduced inflammatory responses in Ubc9 transgenic mice, as key chemotactic signaling molecules such as IL17A were significantly downregulated. Together, our data indicate a critical role particularly of the SUMO2/3 isoforms in modulating responses to I/R and provide the first evidence that SUMO1 deletion activates a compensatory process that protects from ischemic damage.

Scientific hypotheses may either predict particular unknown facts or accommodate previously-known data. Although affirmed predictions are intuitively more rewarding than accommodations of established facts, opinions divide whether predictive hypotheses are also epistemically superior to accommodation hypotheses. This paper examines the contribution of predictive hypotheses to discoveries of several bio-molecular systems. Having all the necessary elements of the system known beforehand, an abstract predictive hypothesis of semiconservative mode of DNA replication was successfully affirmed. However, in defining the genetic code whose biochemical basis was unclear, hypotheses were only partially effective and supplementary experimentation was required for its conclusive definition. Markedly, hypotheses were entirely inept in predicting workings of complex systems that included unknown elements. Thus, hypotheses did not predict the existence and function of mRNA, the multiple unidentified components of the protein biosynthesis machinery, or the manifold unknown constituents of the ubiquitin-proteasome system of protein breakdown. Consequently, because of their inability to envision unknown entities, predictive hypotheses did not contribute to the elucidation of cation theories remained the sole instrument to explain complex bio-molecular systems, the philosophical question of alleged advantage of predictive over accommodative hypotheses became inconsequential.

SIPL1 inhibits PTEN function and stimulates NF-κB signaling; both processes contribute to resistance to hormone therapy in estrogen receptor positive breast cancer (ER+ BC). However, whether SIPL1 promotes tamoxifen resistance in BC remains unclear. We report here that SIPL1 enhances tamoxifen resistance in ER+ BC. Overexpression of SIPL1 in MCF7 and TD47 cells conferred tamoxifen resistance. In MCF7 cell-derived tamoxifen resistant (TAM-R) cells, SIPL1 expression was upregulated and knockdown of SIPL1 in TAM-R cells re-sensitized the cells to tamoxifen. Furthermore, xenograft tumors produced by MCF7 SIPL1 cells but not by MCF7 empty vector cells resisted tamoxifen treatment. Collectively, we demonstrated a role of SIPL1 in promoting tamoxifen resistance in BC. Increases in AKT activation and NF-κB signaling were detected in both MCF7 SIPL1 and TAM-R cells; using specific inhibitors and unique SIPL1 mutants to inhibit either pathway significantly reduced tamoxifen resistance. A SIPL1 mutant defective in activating both pathways was incapable of conferring resistance to tamoxifen, showing that both pathways contributed to SIPL1-derived resistance to tamoxifen in ER+ BCs. Using the Curtis dataset of breast cancer (n=1980) within the cBioPortal database, we examined a correlation of SIPL1 expression with ER+ BC and resistance to hormone therapy. SIPL1 upregulation strongly associates with reductions in overall survival in BC patients, particularly in patients with hormone naïve ER+ BCs. Taken together, we provide data suggesting that SIPL1 contributes to promote resistance to tamoxifen in BC cells through both AKT and NF-κB actions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Interferon-stimulated gene 15 (ISG15) is an important cytokine that has been reported in carcinogenesis. However, we found that ISG15 and de-ISGylase USP18 were induced by several anti-cancer agents, which was confirmed by both RT-PCR and immunoblotting assays. Further studies demonstrated that ectopic ISG15 and USP18 inhibited proliferation of myeloma, leukemia and cervical cancer cells. More importantly, ISG15 and USP18 induced cancer cell apoptosis. This finding was confirmed in a cervical xenograft model in which cervical cancer growth was suppressed by lentiviral ISG15. In the mechanistic study, ISG15 was found to disrupt the NF-κB signaling pathway by downregulating the expression of IKKβ and p65, phosphorylation of p65 and IκBα. Consistent with this finding, ISG15 suppressed the expression of NF-κB recognition element-driving luciferase and decreased the transcription of XIAP and Mcl-1, two typical genes regulated by NF-κB. Therefore, the present study demonstrated that ISG15 induces cancer cell apoptosis by disrupting the NF-κB signaling pathway. This study highlighted a novel role of ISG15 in tumor suppression.

Background: Since innate immunity plays a pivotal role in the pathogenesis of glomerulonephritis, the activation of toll-like receptor (TLR) 3/type I interferon (IFN) cascades is important in glomerular inflammation. However, the role of IFN-stimulated gene 15 (ISG15), a type IFN-dependent transcript, in glomerular inflammation is unclear. We, therefore, examined the role of ISG15 in innate immune reactions induced by TLR3 signaling in cultured human mesangial cells (MCs)., Methods: We treated MCs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the ISG15 expression by reverse transcription-polymerase chain reaction and western blotting. To examine the regulation of ISG15 expression, we subjected MCs to RNA interference (siRNA) against TLR3, IFN-β, ISG56, and melanoma differentiation-associated gene 5 (MDA5)., Results: ISG15 expression induced by poly IC in MCs was inhibited by siRNA against TLR3 and IFN-β, whereas silencing of ISG56 or MDA5 had no effect. A knockdown of ISG15 upregulated the expression of ISG56, MDA5, CXCL10 and phosphorylated signal transducers and activators of transcription protein 1 (P-STAT1), while a knockdown of ubiquitin-like modifier activating enzyme 7, a key enzyme that conjugates ISG15 to target proteins, did not affect the expression. Knockdown of ubiquitin specific protease 18, an ISG15 isopeptidase, also upregulated P-STAT1, ISG56, MDA5 and CXCL10., Conclusion: Since unconjugated free ISG15 negatively regulates the phosphorylation of STAT1 and its downstream reactions, ISG15 dysregulation may be involved in the pathogenesis of glomerular inflammation. We believe that suitable interventions in these innate immune cascades is desirable for the future therapeutic strategies for glomerulonephritis., (© 2016 S. Karger AG, Basel.)

HLA-F adjacent transcript 10 (FAT10) is a cytokine-inducible ubiquitin-like modifier that is highly expressed in the thymus and directly targets FAT10-conjugated proteins for degradation by the proteasome. High expression of FAT10 in the mouse thymus could be assigned to strongly autoimmune regulator-expressing, mature medullary thymic epithelial cells, which play a pivotal role in negative selection of T cells. Also in the human thymus, FAT10 is localized in the medulla but not the cortex. TCR Vβ-segment screening revealed a changed T cell repertoire in FAT10-deficient mice. Analysis of five MHC class I- and II-restricted TCR-transgenic mice demonstrated an altered thymic negative selection in FAT10-deficient mice. Furthermore, the repertoire of peptides eluted from MHC class I molecules was influenced by FAT10 expression. Hence, we identified FAT10 as a novel modifier of thymic Ag presentation and epitope-dependent elimination of self-reactive T cells, which may explain why the fat10 gene could recently be linked to enhanced susceptibility to virus-triggered autoimmune diabetes., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: UBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2, a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS, we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H, P497S, and P506T) in primary neuroglial cultures and in developing neonatal mouse brains., Results: In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread, sustained expression of ubiquilin-2 in brain. In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin, p62/SQSTM, optineurin, and occasionally TDP-43, but were negative for α-synuclein, neurofilament, tau, and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3-4 months, including nonspecific clasping and rotarod deficits., Conclusions: These findings demonstrate that UBQLN2 mutants (P497H, P497S, and P506T) induce proteinopathy and cause behavioral deficits, supporting a "toxic" gain-of-function, which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration.

Ubiquilin 2 (UBQLN2), a member of the ubiquitin-like protein family (ubiquilins), maintains protein homeostasis. Although UBQLN2 has been implicated in the pathogenesis of neurodegenerative diseases, it is also associated with malignant tumors. Therefore, we examined whether UBQLN2 plays a role in human osteosarcoma. The human osteosarcoma cell line MG63 was transfected with UBQLN2 siRNA and cultured under hypoxic conditions. The rat osteosarcoma cell line COS1NR was inoculated into Fischer 344 rats, followed by injection of UBQLN2 siRNA with atelocollagen. An immunohistochemical analysis of UBQLN2 was performed using 34 cases of human high-grade osteosarcomas, and metastasis-free survival was estimated by the Kaplan-Meier method. Silencing of UBQLN2 by siRNA transfection under hypoxia led to activation of JNK and p38, resulting in induction of apoptosis in the osteosarcoma cell line MG63. Injection of UBQLN2 siRNA suppressed tumor growth in the rat osteosarcoma model, followed by apoptosis induction. The immunohistochemical examination revealed that high UBQLN2 expression was significantly associated with the unfavorable metastasis-free survival of osteosarcoma patients. UBQLN2 plays an important role in resistance to hypoxic stress and enhances tumor progression in osteosarcoma. UBQLN2 may be a new molecular target for chemotherapeutics and a useful clinicopathological marker in human osteosarcoma.

It has been reported that FAT10 plays an important role in cell proliferation. Their activity is increased in malignant cells compared to benign cells. However, the clinical and functional significance of FAT10 expression has not been characterized previously in pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to assess FAT10 expression and to explore its contribution to PDAC. Real-time quantitative PCR was performed to examine FAT10 expression in 38 pairs of fresh frozen PDAC tissues and corresponding noncancerous tissues. Using immunohistochemistry, we performed a retrospective study of the FAT10 expression levels on 134 archival PDAC paraffin-embedded samples. The relationship between FAT10 mRNA expression and clinicopathological features was analyzed by appropriate statistics. Kaplan-Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between FAT10 expression and prognosis of PDAC patients. The relative mRNA expression of FAT10 was significantly higher in PDAC tissues than in adjacent noncancerous tissues (P<0.001). By immunohistochemistry, the data revealed that high FAT10 expression was significantly correlated with clinical stage (P<0.001), histological differentiation (P=0.004), and lymph node metastasis (P=0.013). Consistent with these results, we found that high expression of FAT10 was significantly correlated with poor survival in PDAC patients (P<0.001). Furthermore, Cox regression analyses showed that FAT10 expression was an independent predictor of overall survival. In conclusion, this study confirmed the overexpression of FAT10 and its association with tumor progression in PDAC. It also provided the first evidence that FAT10 expression in PDAC was an independent prognostic factor of patients, which might be a potential diagnostic and therapeutic target of PDAC.

The eukaryotic endoplasmic reticulum (ER) maintains protein homeostasis by eliminating unwanted proteins through the evolutionarily conserved ER-associated degradation (ERAD) pathway. During ERAD, maturation-defective and surplus polypeptides are evicted from the ER lumen and/or lipid bilayer through the process of retrotranslocation and ultimately degraded by the proteasome. An integral facet of the ERAD mechanism is the ubiquitin system, composed of the ubiquitin modifier and the factors for assembling, processing and binding ubiquitin chains on conjugated substrates. Beyond simply marking polypeptides for degradation, the ubiquitin system is functionally intertwined with retrotranslocation machinery to transport polypeptides across the ER membrane.

FAT10 (HLA-F-adjacent transcript 10) is an ubiquitin-like modifier, which has been implicated in immune response and cancer development. In particular, the hypothesis of FAT10 as a mediator of tumorigenesis stems from its ability to associate with a spindle checkpoint protein Mad2 during mitosis and cause aneuploidy, a hallmark of cancer cells. Furthermore, FAT10 is overexpressed in several carcinomas types, including that of liver and colon. Nevertheless, direct evidence linking FAT10 to cell malignant transformation and progression is lacking. Here, we demonstrate that high FAT10 expression enhanced the proliferative, invasive, migratory and adhesive functions of the transformed cell line, HCT116. These observations were consistently demonstrated in an immortalized, non-tumorigenic liver cell line NeHepLxHT. Importantly, FAT10 can induce malignant transformation as evidenced from the anchorage-independent growth as well as in vivo tumor-forming abilities of FAT10-overexpressing NeHepLxHT cells, whereas in rapidly proliferating HCT116, increased FAT10 further augmented tumor growth. FAT10 was found to activate nuclear factor-κB (NFκB), which in turn upregulated the chemokine receptors CXCR4 and CXCR7. Importantly, small interfering RNA depletion of CXCR7 and CXCR4 attenuated cell invasion of FAT10-overexpressing cells, indicating that the CXCR4/7 is crucial for the FAT10-dependent malignant phenotypes. Taken together, our data reveal novel functions of FAT10 in malignant transformation and progression, via the NFκB-CXCR4/7 pathway.

Autophagy complements the ubiquitin-proteasome system in mediating protein turnover. Whereas the proteasome degrades individual proteins modified with ubiquitin chains, autophagy degrades many proteins and organelles en masse. Macromolecules destined for autophagic degradation are 'selected' through sequestration within a specialized double-membrane compartment termed the phagophore, the precursor to an autophagosome, and then are hydrolyzed in a lysosome- or vacuole-dependent manner. Notably, a pair of distinctive ubiquitin-like proteins (UBLs), Atg8 and Atg12, regulate degradation by autophagy in unique ways by controlling autophagosome biogenesis and recruitment of specific cargos during selective autophagy. Here we review structural mechanisms underlying the functions and conjugation of these UBLs that are specialized to provide interaction platforms linked to phagophore membranes.

Predicting the success of a species' colonization into a novel environment is routinely considered to be predicated on niche-space similarity and vacancy, as well as propagule pressure. The role genomic variation plays in colonization success (and the interaction with environment) may be suggested, but has not rigorously been documented. To test an hypothesis that previously observed ecotype-specific polymorphisms between anadromous and landlocked alewife (Alosa pseudoharengus) populations are an adaptive response to osmoregulatory challenges rather than a result of allele sampling at founding, we examined multiple anadromous and landlocked (colonized) populations for their allelic profiles at a conserved region (3'-UTR end) of a β-thymosin gene whose protein product plays a central role in the organization of cytoskeleton. The putatively ancestral β-thymosin allele was prevalent in anadromous populations, whereas a newly derived allele was overrepresented in landlocked populations; a third allele was exclusive to the anadromous populations. We also conducted a complementary set of salinity exposure experiments to test osmoregulatory performance of the alewife ecotypes in contrasting saline environments. The pattern of variation and results from these challenges indicate a strong association of β-thymosin with colonization success and a transition from species with an anadromous life history to one with only a freshwater component., (© 2014 Wiley Periodicals, Inc.)

Selective autophagy ensures recognition and removal of various cytosolic cargoes. Hence, aggregated proteins, damaged organelles, or pathogens are enclosed into the double-membrane vesicle, the autophagosome, and delivered to the lysosome for degradation. This process is mediated by selective autophagy receptors, such as p62/SQSTM1. These proteins recognize autophagic cargo and, via binding to small ubiquitin-like modifiers (UBLs)--Atg8/LC3/GABARAPs and ATG5--mediate formation of selective autophagosomes. Recently, it was found that UBLs can directly engage the autophagosome nucleation machinery. Here, we review recent findings on selective autophagy and propose a model for selective autophagosome formation in close proximity to cargo., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Type 1 diabetes, one of two major forms of diabetes, results from the complete destruction of pancreatic beta cells. Viral infection has been suggested to be a trigger of beta cell destruction, the pathogenesis of type 1 diabetes. The aim of this study was to clarify the role of the protein encoded by intherferon stimulated gene (ISG) 15, an antiviral effector, in the development of this clinical entity. We used the mouse beta cell line MIN6 to investigate the role of ISG15 and paid special attention to apoptosis. Although not detected in native MIN6 cells, free ISG15 and ISG15 conjugated proteins were both present in dose-dependently increased amounts following stimulation with interferon alpha. As assessed both by caspase 3/7 activity and an annexin V assay, the percentage of apoptotic MIN6 cells (after exposure to the inflammatory cytokines of interleukin-1beta plus interferon gamma or tumor necrosis factor alpha) was decreased by pretreatment with adenovirus-expressing ISG15 and increased by expressing a short hairpin RNA directed against ISG15. In conclusion, ISG15 has an anti-apoptotic effect on MIN6 cells. Thus, promoting ISG15 expression in the pancreatic beta cells could be a potential therapeutic approach for patients with type 1 diabetes.

c-Myb plays an essential role in regulation of properly balanced hematopoiesis through transcriptional regulation of genes directly controlling cellular processes such as proliferation, differentiation, and apoptosis. The transcriptional activity and protein levels of c-Myb are strictly controlled through post-translational modifications such as phosphorylation, acetylation, ubiquitination, and SUMOylation. Conjugation of small ubiquitin-like modifier (SUMO) proteins has been shown to suppress the transcriptional activity of c-Myb. SUMO-1 modifies c-Myb under physiological conditions, whereas SUMO-2/3 conjugation was reported in cells under stress. Because stress also activates several cellular protein kinases, we investigated whether phosphorylation of c-Myb changes in stressed cells and whether a mutual interplay exists between phosphorylation and SUMOylation of c-Myb. Here we show that several types of environmental stress induce a rapid change in c-Myb phosphorylation. Interestingly, the phosphorylation of Thr(486), located in close proximity to SUMOylation site Lys(499) of c-Myb, is detected preferentially in nonSUMOylated protein and has a negative effect on stress-induced SUMOylation of c-Myb. Stress-activated p38 MAPKs phosphorylate Thr(486) in c-Myb, attenuate its SUMOylation, and increase its proteolytic turnover. Stressed cells expressing a phosphorylation-deficient T486A mutant demonstrate decreased expression of c-Myb target genes Bcl-2 and Bcl-xL and accelerated apoptosis because of increased SUMOylation of the mutant protein. These results suggest that phosphorylation-dependent modulation of c-Myb SUMOylation may be important for proper response of cells to stress. In summary, we have identified a novel regulatory interplay between phosphorylation and SUMOylation of c-Myb that regulates its activity in stressed cells.

Hibernating squirrels slow blood flow to a crawl, but sustain no damage to brain or other tissues. This phenomenon provides an excellent model of natural tolerance to ischemia. Small ubiquitin-like modifier (SUMO) is a 100-residue peptide that modifies other proteins by being attached to the epsilon amino group of specific lysine residues. The discovery of massive SUMOylation (by both SUMO-1 and SUMO-2/3) occurring in the brains of 13-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation torpor had opened the door to the studies on SUMO and ischemic tolerance reviewed here. Ischemic stress was shown to increase the levels of SUMO conjugation, especially SUMO-2/3, mostly during reperfusion in animal models and during restoration of oxygen and glucose in cell culture systems. Over-expression or depletion of SUMOs and/or Ubc9 (the SUMO E2 conjugating enzyme) increases or decreases (respectively) the levels of SUMO conjugates. Elevated global SUMO conjugations were shown to cytoprotect from ischemic insults; conversely, depressed SUMOylation sensitized cells. Global protein conjugation not only by SUMOs, but also by other ubiquitin-like modifiers (ULMs) including NEDD8, ISG15, UFM1 and FUB1 was shown to be significantly increased in the brains of hibernating ground squirrels during torpor. These increases in multiple ULM conjugations may orchestrate the cellular events in hibernating ground squirrels that induce a state of natural tolerance through their multipronged effects. Certain miRNAs such as the miR-200 family and the miR-182 family were shown, at least partly, to control the levels of these ULM conjugations. Lowering the levels of these miRNAs leads to an increase in global SUMOylation/ULM conjugation, thereby providing the tolerance to ischemia. This suggests that these miRNAs may be good targets for therapeutic intervention in stroke.

In previous studies we identified the interferon stimulated gene 15 (ISG15) as a pro-viral host factor in the pathogenesis of hepatitis C virus (HCV) infection. However, the functional link between ISG15 and the HCV replication cycle is not well understood. Aim of the present study was to functionally analyze the role of ISG15 and to identify possible HCV promoting effector molecules. Isg15 suppression was investigated in the murine subgenomic HCV replicon (MH1) transfected with Isg15-specific siRNA and in C57BL/6 mice intravenously injected with lipid nanoparticles (LNP)-formulated siRNA. Interestingly, the LNP-formulated siRNA led to hepatocyte-specific knockdown of Isg15 in vivo, which mediated a hypo-responsiveness to endogenous and exogenous interferon. A label free proteome analysis accompanied by western blot and quantitative RT-PCR techniques led to identification of five candidate proteins (Heterogeneous nuclear ribonucleoprotein A3 (HnrnpA3), Heterogeneous nuclear ribonucleoprotein K (HnrnpK), Hydroxymethylglutaryl-CoA synthase (Hmgcs1), Isocitrate dehydrogenase cytoplasmic (Idh1) and Thioredoxin domain-containing protein 5 (Txndc5)) that are either involved in lipid metabolism or belong to the family of Heterogeneous nuclear ribonucleoprotein (Hnrnp). All candidate proteins are likely to be associated with the HCV replication complex. Furthermore treatment with HnrnpK-specific siRNA directly suppressed HCV replication in vitro. Taken together these data suggest that targeting Isg15 may represent an attractive novel therapeutic option for the treatment of chronic HCV infection.

Type I interferons (IFNs) exert their effects through the induction of hundreds of IFN-stimulated genes (ISGs), many of which function by inhibiting viral replication and modulating immune responses. ISG15, a di-ubiquitin-like protein, is one of the most abundantly induced ISGs and is critical for control of certain viral and bacterial infections. Like ubiquitin, ISG15 is covalently conjugated to target proteins. In addition, free unconjugated ISG15 is present both intra- and extracellularly. Although much remains to be learned about conjugated ISG15, even less is known about the 2 free forms of ISG15. This article focuses on the role that ISG15 plays during the host response to pathogen challenge, in particular on the recent observations describing the immunomodulatory properties of free ISG15 and its potential implication in disease pathogenesis.

Objective: Based on previous data that have linked the small ubiquitin-like modifier-1 (SUMO-1) to the pathogenesis of rheumatoid arthritis (RA), we have investigated the expression of the highly homologous SUMO family members SUMO-2/3 in human RA and in the human tumour necrosis factor α transgenic (hTNFtg) mouse model of RA and studied their role in regulating disease specific matrixmetalloproteinases (MMPs)., Methods: Synovial tissue was obtained from RA and osteoarthritis (OA) patients and used for histological analyses as well as for the isolation of synovial fibroblasts (SFs). The expression of SUMO-2/3 in RA and OA patients as well as in hTNFtg and wild type mice was studied by PCR, western blot and immunostaining. SUMO-2/3 was knocked down using small interfering RNA in SFs, and TNF-α induced MMP production was determined by ELISA. Activation of nuclear factor-κB (NF-κB) was determined by a luciferase activity assay and a transcription factor assay in the presence of the NF-κB inhibitor BAY 11-7082., Results: Expression of SUMO-2 and to a lesser extent of SUMO-3 was higher in RA tissues and RASFs compared with OA controls. Similarly, there was increased expression of SUMO-2 in the synovium and in SFs of hTNFtg mice compared with wild type animals. In vitro, the expression of SUMO-2 but not of SUMO-3 was induced by TNF-α. The knockdown of SUMO-2/3 significantly increased the TNF-α and interleukin (IL)-1β induced expression of MMP-3 and MMP-13, accompanied by increased NF-κB activity. Induction of MMP-3 and MMP-13 was inhibited by blockade of the NF-κB pathway. TNF-α and IL-1β mediated MMP-1 expression was not regulated by SUMO-2/3., Conclusions: Collectively, we show that despite their high homology, SUMO-2/3 are differentially regulated by TNF-α and selectively control TNF-α mediated MMP expression via the NF-κB pathway. Therefore, we hypothesise that SUMO-2 contributes to the specific activation of RASF.

NEDD8 (NEURAL PRECURSOR CELL-EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED PROTEIN8) is an evolutionarily conserved 8-kD protein that is closely related to ubiquitin and that can be conjugated like ubiquitin to specific lysine residues of target proteins in eukaryotes. In contrast to ubiquitin, for which a broad range of substrate proteins are known, only a very limited number of NEDD8 target proteins have been identified to date. Best understood, and also evolutionarily conserved, is the NEDD8 modification (neddylation) of cullins, core subunits of the cullin-RING-type E3 ubiquitin ligases that promote the polyubiquitylation of degradation targets in eukaryotes. Here, we show that Myeloid differentiation factor-2-related lipid-recognition domain protein ML3 is an NEDD8- as well as ubiquitin-modified protein in Arabidopsis (Arabidopsis thaliana) and examine the functional role of ML3 in the plant cell. Our analysis indicates that ML3 resides in the vacuole as well as in endoplasmic reticulum (ER) bodies. ER bodies are Brassicales-specific ER-derived organelles and, similar to other ER body proteins, ML3 orthologs can only be identified in this order of flowering plants. ML3 gene expression is promoted by wounding as well as by the phytohormone jasmonic acid and repressed by ethylene, signals that are known to induce and repress ER body formation, respectively. Furthermore, ML3 protein abundance is dependent on NAI1, a master regulator of ER body formation in Arabidopsis. The regulation of ML3 expression and the localization of ML3 in ER bodies and the vacuole is in agreement with a demonstrated importance of ML3 in the defense to herbivore attack. Here, we extend the spectrum of ML3 biological functions by demonstrating a role in the response to microbial pathogens.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the aggregation of ubiquitinated proteins in affected motor neurons. Recent studies have identified several new molecular constituents of ALS-linked cellular aggregates, including FUS, TDP-43, OPTN, UBQLN2 and the translational product of intronic repeats in the gene C9ORF72. Mutations in the genes encoding these proteins are found in a subgroup of ALS patients and segregate with disease in familial cases, indicating a causal relationship with disease pathogenesis. Furthermore, these proteins are often detected in aggregates of non-mutation carriers and those observed in other neurodegenerative disorders, supporting a widespread role in neuronal degeneration. The molecular characteristics and distribution of different types of protein aggregates in ALS can be linked to specific genetic alterations and shows a remarkable overlap hinting at a convergence of underlying cellular processes and pathological effects. Thus far, self-aggregating properties of prion-like domains, altered RNA granule formation and dysfunction of the protein quality control system have been suggested to contribute to protein aggregation in ALS. The precise pathological effects of protein aggregation remain largely unknown, but experimental evidence hints at both gain- and loss-of-function mechanisms. Here, we discuss recent advances in our understanding of the molecular make-up, formation, and mechanism-of-action of protein aggregates in ALS. Further insight into protein aggregation will not only deepen our understanding of ALS pathogenesis but also may provide novel avenues for therapeutic intervention.

A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective therapies. We sought to define the most proximal regulators of the Cullin (Cul)-RING ligases, which play a central role in the stabilization of NF-κB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-α by assessing Cul neddylation status, NF-κB and HIF-1α stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α, and increased NF-κB/HIF-α promoter activity in response to LPS. HMECs lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-κB or HIF-1α. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-κB responses, induced HIF-1α promoter activity, and reduced secretion of TNF-α-elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation.

This review highlights the finding that ubiquitin-like (Ubl) proteins of archaea (termed SAMPs) function not only as sulfur carriers but also as protein modifiers. UbaA (an E1 ubiquitin-activating enzyme homolog of archaea) is required for the SAMPs to be covalently attached to proteins. The SAMPs and UbaA are also needed to form sulfur-containing biomolecules (e.g., thiolated tRNA and molybdenum cofactor). These findings provide a new perspective on how Ubl proteins can serve as both sulfur carriers and protein modifiers in the absence of canonical E2 ubiquitin conjugating or E3 ubiquitin ligase enzyme homologs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

IFNs are cytokines that segregate viral infections, modulate the immune responses and influence tumor cells survival. These options are under the control of ISGs (Interferon Stimulated Genes) which expression is propelled by IFNs. To the ISGs family belong all the components of the molecular machinery that modifies proteins by the addition of the ubiquitin-like protein ISG15, in a process known as ISGylation. Despite alterations in the components of this machinery are frequently observed in cancer, the contribution of ISG15 and of ISGylation to tumor growth and resistance to chemotherapy is unclear and debated. With the aim of elucidating this point, in this review we have discussed about recent data pointing to a dysregulation of the IFN signaling and the ISGylation system in cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mallory-Denk bodies (MDBs) are aggresomes composed of undigested ubiqutinated short lived proteins which have accumulated because of a decrease in the rate of their degradation by the 26s proteasome. The decrease in the activity of the proteasome is due to a shift in the activity of the 26s proteasome to the immunoproteasome triggered by an increase in expression of the catalytic subunits of the immunoproteasome which replaces the catalytic subunits of the 26s proteasome. This switch in the type of proteasome in liver cells is triggered by the binding of IFNγ to the IFNγ sequence response element (ISRE) located on the FAT10 promoter. To determine if either FAT10 or IFNγ are essential for the formation of MDBs we fed both IFNγ and FAT10 knock out (KO) mice DDC added to the control diet for 10weeks in order to induce MDBs. Mice fed the control diet and Wild type mice fed the DDC or control diet were compared. MDBs were located by immunofluorescent double stains using antibodies to ubiquitin to stain MDBs and FAT10 to localize the increased expression of FAT10 in MDB forming hepatocytes. We found that MDB formation occurred in the IFNγ KO mice but not in the FAT10 KO mice. Western blots showed an increase in the ubiquitin smears and decreases β 5 (chymotrypsin-like 26S proteasome subunit) in the Wild type mice fed DDC but not in the FAT10 KO mice fed DDC. To conclude, we have demonstrated that FAT10 is essential to the induction of MDB formation in the DDC fed mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Axon degeneration is a characteristic event in many neurodegenerative conditions including stroke, glaucoma, and motor neuropathies. However, the molecular pathways that regulate this process remain unclear. Axon loss in chronic neurodegenerative diseases share many morphological features with those in acute injuries, and expression of the Wallerian degeneration slow (WldS) transgene delays nerve degeneration in both events, indicating a common mechanism of axonal self-destruction in traumatic injuries and degenerative diseases. A proposed model of axon degeneration is that nerve insults lead to impaired delivery or expression of a local axonal survival factor, which results in increased intra-axonal calcium levels and calcium-dependent cytoskeletal breakdown.

ISG15 is a 15kD ubiquitin-like protein (UBL) induced by interferon (IFN). ISG15 can be covalently attached to proteins, which is called ISGylation process. ISGylation system contains ISG15, UBE1L, UBCH8 and HERC5 proteins, which are all essential for ISGylation. ISG15 and ISGylation system have been found to have anti-viral effects. A better understanding of how ISG15 mediates the anti-viral activity will provide insights for new anti-viral drugs development and new therapeutic strategies. The mechanisms underlying the ISG15 mediated anti-viral response have been explored extensively in recent years. This minireview summarized the research advances of how ISG15 mediated the anti-viral effects against different kinds of viruses.

Ubiquitin (Ub) and ubiquitin-like (UBL) proteins regulate a diverse array of cellular pathways through covalent as well as non-covalent interactions with target proteins. Yeast protein Mdy2 (Get5) and its human homolog GdX (Ubl4a) belong to the class of UBL proteins which do not form conjugates with other proteins. Mdy2 is required for cell survival under heat stress and for efficient mating. As part of a complex with Sgt2 and Get4 it has been implicated in the biogenesis of tail-anchored proteins. Interestingly, in response to heat stress, Mdy2 protein that is predominantly localized in the nucleus co-localized with poly(A)-binding protein Pab1 to cytoplasmic stress granules suggesting that nucleocytoplasmic shuttling is of functional importance. Here we investigate the nuclear import of Mdy2, a process that is independent of the Get4/Sgt2 complex but required for stress response. Nuclear import is mediated by an N-terminal nuclear localization signal (NLS) and this process is essential for the heat stress response. In contrast, cells expressing Mdy2 lacking a nuclear export signal (NES) behave like wild type. Importantly, both Mdy2 and Mdy2-ΔNES, but not Mdy2-ΔNLS, physically interact with Pab1 and this interaction correlates with the accumulation in cytoplasmic stress granules. Thus, the nuclear history of the UBL Mdy2 appears to be essential for its function in cytoplasmic stress granules during the rapid cellular response to heat stress.