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During mammalian meiosis, Polo-like kinase 1 (PLK1) is essential during cell cycle progression. In oocyte maturation, PLK1 expression is well characterized but timing of posttranslational modifications regulating its activity and subcellular localization are less clear. Small ubiquitin-related modifier (SUMO) posttranslational modifier proteins have been detected in mammalian gametes but their precise function during gametogenesis is largely unknown. In the present paper we report for mouse oocytes that both PLK1 and phosphorylated PLK1 undergo SUMOylation in meiosis II (MII) oocytes using immunocytochemistry, immunoprecipitation and in vitro SUMOylation assays. At MII, PLK1 is phosphorylated at threonine-210 and serine-137. MII oocyte PLK1 and phosphorylated PLK1 undergo SUMOylation by SUMO-1, −2 and −3 as shown by individual in vitro assays. Using these assays, forms of phosphorylated PLK1 normalized to PLK1 increased significantly and correlated with SUMOylated PLK1 levels. During meiotic progression and maturation, SUMO-1-SUMOylation of PLK1 is involved in spindle formation whereas SUMO-2/3-SUMOylation may regulate PLK1 activity at kinetochore-spindle attachment sites. Microtubule integrity is required for PLK1 localization with SUMO-1 but not with SUMO-2/3. Inhibition of SUMOylation disrupts proper meiotic bipolar spindle organization and spindle-kinetochore attachment. The data show that both temporal and SUMO-specific-SUMOylation play important roles in orchestrating functional dynamics of PLK1 during mouse oocyte meiosis, including subcellular compartmentalization. [ABSTRACT FROM AUTHOR]

Small ubiquitin-related modifiers (SUMOs) conjugated or bound to target proteins can affect protein trafficking, processing and solubility. SUMOylation has been suggested to play a role in the amyloid plaque and neurofibrillary tangle pathology of Alzheimer disease (AD) and related neurodegenerative diseases. The current study examines the impact of SUMO1 on processing of the amyloid precursor protein (APP) leading to the production and deposition of the amyloid-β (Aβ) peptide. An in vivo model of these pathways was developed by the generation of double transgenic mice over-expressing human SUMO1 and a mutant APP. The SUMO1-APP transgenics displayed normal APP processing but, at later ages, exhibited increased insoluble Aβ and plaque density accompanied by increased dendritic spine loss, more pronounced synaptic and cognitive deficits. These findings suggest a potential impairment in Aβ clearance as opposed to increased amyloid production. Examination of microglia indicated a reduction in the SUMO1-APP transgenics which is a possible mechanism for the SUMO1-mediated increase in amyloid load. These findings suggest an indirect activity of SUMO1 possibly in the removal of Aβ plaques rather than a direct impact on amyloid generation. [ABSTRACT FROM AUTHOR]

The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation. [ABSTRACT FROM AUTHOR]

Posttranslational modification by small ubiquitin-like modifier (SUMO) regulates myriad physiological processes within cells and has been demonstrated to be highly activated in murine brains after cerebral ischemia. Numerous in vitro and murine in vivo studies have demonstrated that this increased SUMO conjugation is an endogenous neuroprotective stress response that has potential in being leveraged to develop novel therapies for ischemic stroke. However, SUMO activation has not yet been studied in poststroke human brains, presenting a clear limitation in translating experimental successes in murine models to human patients. Accordingly, here, we present a case wherein the brain tissue of a stroke patient (procured shortly after death) was processed by multiplex immunohistochemistry to investigate SUMO activation. [ABSTRACT FROM AUTHOR]

Histone exchange and histone post-translational modifications play important roles in the regulation of DNA metabolism, by re-organizing the chromatin configuration. We previously demonstrated that the histone variant H2A.Z-2 is rapidly exchanged at damaged sites after DNA double strand break induction in human cells. In yeast, the small ubiquitin-like modifier (SUMO) modification of H2A.Z is involved in the DNA damage response. However, whether the SUMO modification regulates the exchange of human H2A.Z-2 at DNA damage sites remains unclear. Here, we show that H2A.Z-2 is SUMOylated in a damage-dependent manner, and the SUMOylation of H2A.Z-2 is suppressed by the depletion of the SUMO E3 ligase, PIAS4. Moreover, PIAS4 depletion represses the incorporation and eviction of H2A.Z-2 at damaged sites. These findings demonstrate that the PIAS4-mediated SUMOylation regulates the exchange of H2A.Z-2 at DNA damage sites. [ABSTRACT FROM AUTHOR]

Conjugation of SUMO (Small Ubiquitin-like Modifier) protein to cellular targets is emerging as a very influential protein modification system. Once covalently bound, SUMO conjugation can change the stability or functionality of its cognate target proteins. SUMO protease can rapidly reverse SUMO conjugation making this modification system highly dynamic. A major factor in the variation of SUMO-target function is the balance between the conjugated/de-conjugated forms. The mechanistic role of these regulatory SUMO proteases in mediating stress responses has not been defined in any crops. In this study, we reveal the role of the SUMO protease, Os OTS1 in mediating tolerance to drought in rice. Os OTS1 depleted transgenic plants accumulate more ABA and exhibit more productive agronomic traits during drought while Os OTS1 overexpressing lines are drought sensitive but ABA insensitive. Drought and ABA treatment stimulates the degradation of Os OTS1 protein indicating that SUMO conjugation is an important response to drought stress in rice achieved through down-regulation of OTS1/2 activity. We reveal that Os OTS1 SUMO protease directly targets the ABA and drought responsive transcription factor Osb ZIP23 for de- SUMOylation affecting its stability. Os OTS- RNAi lines show increased abundance of Osb ZIP23 and increased drought responsive gene expression while Os OTS1 overexpressing lines show reduced levels of Osb ZIP23 leading to suppressed drought responsive gene expression. Our data reveal a mechanism in which rice plants govern ABA-dependant drought responsive gene expression by controlling the stability of Osb ZIP23 by SUMO conjugation through manipulating specific SUMO protease levels. [ABSTRACT FROM AUTHOR]

Heparinase I (Hep I) can efficiently depolymerize heparin and heparin sulfate to oligosaccharides or unsaturated disaccharides, which resulted in loss of physiological function such as blood coagulation. In order to realize the immobilization of Hep I on chitin carriers, we cloned Hep I with the chitin binding domain (ChBD) as a chitin-affinity tag, and the Small Ubiquitin-like MOdifier (SUMO) linker as a solvation enhancer in different fusion sequence. DNA and protein gels suggested that 4 kinds of recombinants were successfully constructed and expressed in Escherichia coli ( E. coli ). And the triple functional heparinases isolated from cell lysate could be efficiently purified by chitin beads. After optimizing fermentation conditions, it gave the specific enzyme activities of 1.88 ± 0.11, 3.69 ± 0.45, 3.44 ± 0.38, and 2.73 ± 0.29 IU/mg total proteins for ChBD-Hep I, ChBD-SUMO-Hep I, SUMO-ChBD-Hep I, and ChBD-Hep I-SUMO, respectively, with unfractionated heparin as substrate. The optimal reaction temperature and pH were determined to be 30 °C and 7.0 for all the fusion enzymes. ChBD-SUMO-Hep I exhibited the maximum half-life (48 min) at 30 °C and best thermo-stability under 15–50 °C. All the fusion enzymes showed broad pH-stability in the range of 5.4–9.0. [ABSTRACT FROM AUTHOR]

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase. [ABSTRACT FROM AUTHOR]

In Arabidopsis more than 400 proteins have been identified as SUMO targets, both in vivo and in vitro. Among others, transcription factors (TFs) are common targets for SUMO conjugation. Here we aimed to exhaustively screen for TFs that interact with the SUMO machinery using an arrayed yeast two-hybrid library containing more than 1,100 TFs. We identified 76 interactors that foremost interact with the SUMO conjugation enzyme SCE1 and/or the SUMO E3 ligase SIZ1. These interactors belong to various TF families, which control a wide range of processes in plant development and stress signaling. Amongst these interactors, the TCP family was overrepresented with several TCPs interacting with different proteins of the SUMO conjugation cycle. For a subset of these TCPs we confirmed that the catalytic site of SCE1 is essential for this interaction. In agreement, TCP1, TCP3, TCP8, TCP14, and TCP15 were readily SUMO modified in an E. coli sumoylation assay. Strikingly, these TCP-SCE1 interactions were found to redistribute these TCPs into nuclear foci/speckles, suggesting that these TCP foci represent sites for SUMO (conjugation) activity. [ABSTRACT FROM AUTHOR]

Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1–mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions. [ABSTRACT FROM AUTHOR]

Acute cerebral infarction can progress rapidly, and there are limited specific and effective treatments. Small ubiquitin‑like modifiers (SUMOs) provide an important post‑translational modification of proteins. Following cerebral infarction, multiple proteins can combine with SUMOs to protect nerve cells. Furthermore, moderate hypothermia (core body temperature, 33‑34˚C) can increase the level of SUMOylation on multiple proteins. In the present study, it was examined whether moderate hypothermia increases the survival rate of bone marrow stromal stem cells (BMSCs) implanted in the cerebral ischemic penumbra via SUMOylation of multiple proteins. Firstly, BMSCs were exposed to oxygen‑glucose deprivation (OGD) under moderate hypothermic (33˚C) conditions. Subsequently, adult rats with middle cerebral artery occlusion were treated with a combination of BMSCs and moderate hypothermia (32‑34˚C). The results demonstrated that hypothermia promoted the combination of multiple proteins with SUMOs in BMSCs, and induced transport of SUMOs from the cytoplasm to the nucleus. Moderate hypothermia additionally reduced damage to BMSCs following OGD and improved BMSC survival following transplantation into the penumbra. These data suggest that moderate hypothermia may protect against BMSC injury via rapid SUMOylation of intracellular proteins. Thus, BMSC transplantation combined with moderate hypothermia may be a potential therapeutic strategy to treat cerebral infarction. [ABSTRACT FROM AUTHOR]

Small ubiquitin-like modifier (SUMO) proteins belong to the ubiquitin-like family and act to change the function of target proteins through post-translational modifications. Through their interactions with innate immune pathways, SUMOs promote an efficient immune response to pathogenic challenge avoiding, at the same time, an excess of immune response that could lead to the development of autoimmune diseases. This report discusses the general functions of SUMO proteins; highlights SUMO involvement in the innate immune response through their role in NF-κB and interferon pathways; the involvement of SUMO proteins in autoimmune diseases; and reviews bacterial, viral, and parasitic interactions with SUMO pathways. In conclusion, we speculate that targeting SUMOs could represent a new therapeutic strategy against infections and autoimmunity. [ABSTRACT FROM AUTHOR]

DNA Topoisomerase IIα (Topo IIα) is a ubiquitous enzyme in eukaryotes that performs the strand passage reaction where a double helix of DNA is passed through a second double helix. This unique reaction is critical for numerous cellular processes. However, the enzyme also possesses a C-terminal domain (CTD) that is largely dispensable for the strand passage reaction but is nevertheless important for the fidelity of cell division. Recent studies have expanded our understanding of the roles of the Topo IIα CTD, in particular in mitotic mechanisms where the CTD is modified by Small Ubiquitin-like Modifier (SUMO), which in turn provides binding sites for key regulators of mitosis. [ABSTRACT FROM AUTHOR]

Menin, encoded by the multiple endocrine neoplasia type 1 ( MEN1 ) gene, is a tumor suppressor and transcription regulator. Menin interacts with various proteins as a scaffold protein and is proposed to play important roles in multiple physiological and pathological processes by controlling gene expression, proliferation, and apoptosis. The mechanisms underlying menin's suppression of tumorigenesis are largely elusive. In this study, we showed that menin was essential for the regulation of canonical Wnt/β-catenin signaling in cultured cells. The C-terminal domain of menin was able to directly interact with and promote ubiquitin-mediated degradation of β-catenin. We further revealed that overexpression of menin down-regulated the transcriptional activity of β-catenin and target gene expression. Moreover, menin efficiently inhibited β-catenin protein levels, transcriptional activity, and proliferation of human renal carcinoma cells with an activated β-catenin pathway. Taken together, our results provide novel molecular insights into the tumor suppressor activity of menin, which is partly mediated by proteasomal degradation of β-catenin and inhibition of Wnt/β-catenin signaling. [ABSTRACT FROM AUTHOR]

The response to a DNA double-stranded break inmammalian cells is a process of sensing and signalling the lesion. It results in halting the cell cycle and local transcription and in the mediation of the DNA repair process itself. The response is launched through a series of post-translational modification signalling events coordinated by phosphorylation and ubiquitination. More recently modifications of proteins by Small Ubiquitin-like MOdifier (SUMO) isoforms have also been found to be key to coordination of the response (Morris et al. 2009 Nature 462, 886-890 (doi:10.1038/nature08593); Galanty et al. 2009 Nature 462, 935-939 (doi:10.1038/nature08657)). However our understanding of the role of SUMOylation is slight compared with our growing knowledge of how ubiquitin drives signal amplification and key chromatin interactions. In this review we consider our current knowledge of how SUMO isoforms, SUMO conjugation machinery, SUMO proteases and SUMO-interacting proteins contribute to directing altered chromatin states and to repair-protein kinetics at a double-stranded DNA lesion in mammalian cells. We also consider the gaps in our understanding. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) replication in cells is controlled by many host factors. In this report, we found that protein inhibitor of activated STAT2 (PIAS2), which is a small ubiquitin-like modifier (SUMO) E3 ligase, restricted HCV replication. During infection, HCV core, NS3 and NS5A protein expression, as well as the viral assembly and budding efficiency were enhanced when endogenous PIAS2 was knocked down, whereas exogenous PIAS2 expression decreased HCV core, NS3, and NS5A protein expression and the viral assembly and budding efficiency. PIAS2 did not influence the viral entry, RNA replication, and protein translation steps of the viral life cycle. When expressed together with SUMO1, PIAS2 reduced the HCV core, NS3 and NS5A protein levels expressed from individual plasmids through the proteasome pathway in a ubiquitin-independent manner; the stability of these proteins in the HCV infectious system was enhanced when PIAS2 was knocked down. Furthermore, we found that the core was SUMOylated at amino acid K78, and PIAS2 enhanced the SUMOylation level of the core. [ABSTRACT FROM AUTHOR]

MdMYB1 acts as a crucial component of the MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis in red-skinned apples ( Malus domestica), but little is known about its post-translational regulation. Here, a small ubiquitin-like modifier E3 ligase MdSIZ1 was screened out as an MdMYB1-interacting protein with a yeast two-hybridization approach. The interaction between MdSIZ1 and MdMYB1 was further verified with pull-down and CoIP assays. Furthermore, it was found that MdSIZ1 directly sumoylated MdMYB1 proteins in vivo and in vitro, especially under moderately low temperature (17 °C) conditions, and that this sumoylation was required for MdMYB1 protein stability. Moreover, the transcription level of MdSIZ1 gene was remarkably induced by low temperature and phosphorus deficiency, and MdSIZ1 overexpression exerted a large positive influence on anthocyanin accumulation and red fruit coloration, suggesting its important role in the regulation of anthocyanin biosynthesis under stress conditions. Our findings reveal an important role for a small ubiquitin-like modifier modification of MYB transcription factors in regulation of anthocyanin biosynthesis in plants. [ABSTRACT FROM AUTHOR]

Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear.We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a “split-SIM” SUMO2 engagement platform. These findings uncover a ZATT-TDP2–catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc. [ABSTRACT FROM AUTHOR]

How hypothermia serves an early protective role against cerebral ischemia remains to be determined. The small ubiquitin‑related modifier protein (SUMO) functions as a post‑translational modification system and SUMO‑2/3 subtypes are often activated in early stress. The present study investigated changes in the protein level of SUMO using western blotting and immunocytochemistry when neurons were exposed to oxygen‑glucose deprivation (OGD) in vitro, as well as in a rat model of middle cerebral artery occlusion (MCAO) in vivo. The results demonstrated that a large number of proteins were conjugated to SUMO‑2/3 in OGD‑injured neurons (within 10 min, peaking at 12 h), and was markedly enhanced under conditions of hypothermia (33˚C). Concordantly, lactate dehydrogenase (LDH) release and the apoptosis rate, as determined by LDH and TUNEL assays, respectively, were lower in hypothermia‑treated neurons. Similar results were obtained in a rat model of MCAO. Neurological deficit scores were lower in the hypothermia group than in the sham group in the early stage of cerebral ischemia (P<0.05). However, no significant differences in neurological deficit scores were detected between the hypothermia group and the sham group in the late stage of ischemia (21 days; P>0.05). This study implicates a role for SUMO‑2/3 in early hypothermia‑induced neuroprotection against stroke. The development of small molecule therapeutics based on SUMO‑2/3 may benefit patients with cerebral ischemia. [ABSTRACT FROM AUTHOR]

Many nascent proteins are assembled into multiprotein complexes of defined stoichiometry. Imbalances in the synthesis of individual subunits result in orphans. Howorphans are selectively eliminated to maintain protein homeostasis is poorly understood. Here, we found that the conserved ubiquitin-conjugating enzyme UBE2O directly recognized juxtaposed basic and hydrophobic patches on unassembled proteins to mediate ubiquitination without a separate ubiquitin ligase. In reticulocytes, where UBE2O is highly up-regulated, unassembled a-globin molecules that failed to assemble with b-globin were selectively ubiquitinated by UBE2O. In nonreticulocytes, ribosomal proteins that did not engage nuclear import factorswere targets for UBE2O. Thus, UBE2O is a self-contained quality control factor that comprises substrate recognition and ubiquitin transfer activities within a single protein to efficiently target orphans of multiprotein complexes for degradation. [ABSTRACT FROM AUTHOR]

Conjugation of the small ubiquitin-related modifier ( SUMO) to protein substrates has an impact on stress responses and on development. We analyzed the proteome and phosphoproteome of mutants in this pathway. The mutants chosen had defects in SUMO ligase SIZ1, which catalyzes attachment of single SUMO moieties onto substrates, and in ligases PIAL1 and PIAL2, which are known to form SUMO chains. A total of 2657 proteins and 550 phosphopeptides were identified and quantified. Approximately 40% of the proteins and 20% of the phosphopeptides showed differences in abundance in at least one of the analyzed genotypes, demonstrating the influence of SUMO conjugation on protein abundance and phosphorylation. The data show that PIAL1 and PIAL2 are integral parts of the SUMO conjugation system with an impact on stress response, and confirm the involvement of SIZ1 in plant defense. We find a high abundance of predicted SUMO attachment sites in phosphoproteins (70% versus 40% in the total proteome), suggesting convergence of phosphorylation and sumoylation signals onto a set of common targets. [ABSTRACT FROM AUTHOR]

Viruses exploit various cellular processes for their own benefit, including counteracting anti-viral responses and regulating viral replication and propagation. In the past 20 years, protein sumoylation has emerged as an important post-translational modification that is manipulated by viruses to modulate anti-viral responses, viral replication, and viral pathogenesis. The process of sumoylation is a multi-step cascade where a small ubiquitin-like modifier (SUMO) is covalently attached to a conserved ΨKxD/E motif within a target protein, altering the function of the modified protein. Here we review how viruses manipulate the cellular machinery at each step of the sumoylation process to favor viral survival and pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Chromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. Results: Here we addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. Conclusions: These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation. [ABSTRACT FROM AUTHOR]

Aims The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase® cultivation mode and a redox mutant strain in the E. coli expression system. Methods and Results A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis®. Conclusions This study demonstrated that the combination of SUMO fusion technology, EnBase® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. Significance and the Impact of the study The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. [ABSTRACT FROM AUTHOR]

The process of protein modification by adding or detaching small ubiquitin-like modifiers (SUMO) proteins, called SUMOylation, contributes to the regulation of numerous processes in eukaryotic cells. SUMOylation also represents a key response and adaption mechanism to different forms of metabolic stress. The central nervous system (CNS) and neurons in particular are highly susceptible to hypoxic-ischemic stress due to the lack of significant oxygen and energy reserves. SUMOylation is observed in many molecular responses to metabolic stress in the brain, and is therefore supposed to represent an endogenous neuroprotective mechanism. However, the detailed roles of SUMOylation during CNS hypoxia-ischemia are not well understood so far. Moreover, SUMOylation is subjected to complex regulatory mechanisms and might exert protective, but also detrimental processes during hypoxic-ischemic stress. This review provides a comprehensive overview on SUMOylation processes under physiological and pathological conditions in the CNS. A particular spotlight is set on clinically relevant hypoxic-ischemic conditions such as stroke by focusing on peri- and postischemic SUMOylation in neurons and astrocytes. The review describes relevant SUMOylation targets in these cells to discuss confirmed and supposed downstream mechanisms potentially contributing to neuroprotection, but also to sometimes detrimental processes. The review further provides unique insights into the time course of SUMO responses during cerebral ischemia in different cerebral cell populations. This includes neurons, astrocytes, but also phagocytes that become activated (microglia) and/or migrate (macrophages/monocytes) to the ischemic CNS. Based on this compact knowledge, the review finally suggests potential directions for future basic and translational research. [ABSTRACT FROM AUTHOR]

Disruption of the neurovascular unit (NVU), induced by chronic cerebral hypoperfusion (CCH), has been broadly found in various neurological disorders. SUMO-specific protease 3 (SENP3) is expressed in neurons, astrocytes, and microglia, and regulates a variety of cell events. However, whether SENP3 is involved in neurovascular injury under the condition of CCH is still elusive. To address this issue, we investigated the effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on NVU and the role of SENP3 in this process, as well as the underling mechanisms. The expression of SENP3 was detected by immunochemistry. The function and structure of the NVU was assessed by Western blot analysis and transmission electron microscopy. CCH caused the upregulation of SENP3, the disruption of cell and non-cell components at the protein level within the NVU, and ultrastructural deterioration. The NVU impairment as well as overexpression of SENP3 were reversed by treatment with URB597. These results reveal a novel neuroprotective role in URB597, which implicates URB597 in the amelioration of CCH-induced NVU impairment by inhibiting SENP3. [ABSTRACT FROM AUTHOR]

The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites. [ABSTRACT FROM AUTHOR]

Small Ubiquitin-related MOdifier (SUMO) proteins can be found in many organisms, including A. thaliana, which possesses 9 SUMO genes. SUMO binds to various target proteins in a reversible reaction called SUMOylation. SUMOylation participates in transcription, chromosome organization, proteins localizations and stress responses. Our study showed that RIN13 (RPM1-Interacting13/At2g20310) is a target of SUMOylation, which was initially found by interaction between this protein and AtSCE1a (E2). Recent report showed that overexpression of RIN13 enhanced the resistance to pathogen without inducing hypersensitive response. However, the molecular interaction between RIN13 and SUMO proteins and its significance have not been studied yet. Thus, our study aimed to characterize the Interaction between RIN13 and SUMO proteins in A. thaliana. The result showed an isoform-specific SUMOylation between RIN13 and SUMO proteins. RIN13 is SUMOylated by SUMO1, 2, 3 and 5. Though expressed ubiquitously in A.thaliana, fluorescence microscopy showed that RIN13 localizes subcellularly in the nuclear body. Moreover, complete abolishment of SUMOylation with inactive E2 suggests the exclusion of RIN13 from nuclear body. These results showed that SUMOylation affected RIN13 localization and indirectly influenced its interaction to other proteins and putative function. This paper presents evidence of RIN13 SUMOylation. Furthermore, RIN13 function in pathogenic resistance is shown to be supported by SUMOylation. Thus, this study enhanced the understanding of SUMO in plants and served as reference to molecular studies concerning post-translational modification of SUMO. [ABSTRACT FROM AUTHOR]

Objective: To fabricate an active and stable enzyme through covalent immobilization, a Ubl-specific protease (Ulp1) was used to cleave small ubiquitin-like modifier (SUMO) fusion proteins. Results: We immobilized Ulp1 on N-hydroxysuccinimide (NHS)-activated Sepharose with a coupling efficiency of 1.7 mg/ml. The immobilized Ulp1 maintains 95% substrate-cleavage ability and significantly enhances pH and thermal stability, especially can withstand pH of 10.5. Besides resistance against some small molecules, the immobilized Ulp1 can tolerate 15% (v/v) DMSO and 20% (v/v) ethanol. It can be reused for more than 15 batch reactions with 90% activity retention. This provides a fast purification system to quickly obtain cleaved recombinant proteins with 95% purity from cell lysates with the application of immobilized Ulp1. Conclusions: Ulp1 used in immobilization form is a potentially useful tool for cleavage of SUMO-tagged proteins and may reduce time and cost of protein purification. [ABSTRACT FROM AUTHOR]

The present study investigated the protective effects of small ubiquitin‑like modifier 1 (SUMO‑1) on spinal cord ischemic damage in rabbits. A trans‑activator of transcription (Tat)‑SUMO‑1 fusion protein was prepared, and transient spinal cord ischemia was induced by occlusion of the abdominal aorta for 15 min. Vehicle (glycerol) or 1 mg/kg Tat‑1‑SUMO‑1 was administered intraperitoneally to the rabbits immediately following ischemia/reperfusion. Administration of Tat‑SUMO‑1 did not lead to significant alterations in arterial blood gases [partial pressure (Pa)CO2 and PaO2], pH, or blood glucose levels prior to ischemia, 10 min after occlusion or 10 min after reperfusion. Mean arterial pressure was significantly decreased only during occlusion. Motor behaviors were assessed at 24, 48 and 72 h after ischemia/reperfusion using Tarlov's criteria. Administration of Tat‑SUMO‑1 significantly improved Tarlov scores 24 h after ischemia/reperfusion and the number of cresyl violet positive neurons was significantly increased in the ventral horn of the spinal cord compared with the vehicle‑treated group. However, Tarlov scores were consistently decreased at 48 and 72 h after ischemia/reperfusion in the Tat‑SUMO‑1‑treated group, and Tarlov scores and the number of cresyl violet positive neurons were not significantly different between the vehicle- and Tat‑SUMO‑1‑treated groups after 72 h. Tat‑SUMO‑1 administration significantly ameliorated a reduction in Cu, Zn‑superoxide dismutase activity and an increase in lipid peroxidation 24 h after ischemia/reperfusion; however, these effects were not present at 72 h. These results suggested that Tat‑SUMO‑1 may delay, although not protect against, neuronal death by regulating oxidative stress in the ventral horn of the spinal cord and that combination therapy using Tat‑SUMO‑1 with other compounds may provide a therapeutic approach to decrease neuronal damage. [ABSTRACT FROM AUTHOR]

Previously, we reported that small ubiquitin-like modifier-1 (SUMO-1) promotes the degradation of a polyglutamine (polyQ) protein ataxin-3 and proposed that proteasomes mediate the proteolysis. Here, we present evidence that autophagy is also responsible for SUMO-induced degradation of this polyQ protein. The autophagy inhibitor 3-MA increased the steady-state level of ataxin-3 and stabilized SUMO-modified ataxin-3 more prominently than the proteasome inhibitor MG132. Interestingly, SUMO-1 overexpression enhanced the co-localization of ataxin-3 and autophagy marker LC3 without increasing LC3 puncta formation suggesting that SUMO-1 is involved in the substrate recruitment rather than the induction of autophagy. To assess the importance of a putative SUMO-interacting motif (SIM) in ataxin-3 for SUMO-induced degradation, we constructed a SIM mutant of ataxin-3. Substitution of putative SIM (V165G) facilitated the degradation of polyQ-expanded ataxin-3, which is more resistant to SUMO-induced degradation than the normal ataxin-3. These results together indicate that SUMO-1 promotes the degradation of ataxin-3 via autophagy and the putative SIM of ataxin-3 plays a role in this process. [ABSTRACT FROM PUBLISHER]

The article focuses on a study related to association of myositis-specific antibodies (MSAs) in a dermatomyositis (DM) conditions. Topics discussed include characterization of MSAs in characterization for remodelling enzymes such as anti-transcription factor-1 (Anti-TIF-1); performance of different diagnostic services among patients such as serology; and examination of cutaneous phenotypes occurrence as calcinosis in serotypes of MSA.

The family of RecQ helicases is evolutionary conserved from bacteria to humans and play key roles in genome stability. The budding yeast RecQ helicase Sgs1 has been implicated in several key processes during the repair of DNA damage by homologous recombination as part of the STR complex (Sgs1-Top3-Rmi1). Limited information on how is Sgs1 recruited and regulated at sites of damage is available. Recently, we and others have uncover a direct link between the Smc5/6 complex and Sgs1. Most roles of Sgs1 during recombination, including DNA end resection, Holiday junction dissolution, and crossover suppression, are regulated through Mms21-dependent SUMOylation. Smc5/6 first acts as a recruiting platform for STR and then SUMOylates STR components to regulate their function. Importantly, the assembly of STR is totally independent of Smc5/6. Here, we provide a brief overview of STR regulation by Smc5/6. [ABSTRACT FROM AUTHOR]

Background: It has been reported that blocking small ubiquitin-like modifier (SUMO) conjugation by silencing SUMO gene remarkably decreased tumor growth in vivo. However, few studies have examined the relationship between SUMO gene silencing and gastric cancer (GC). The study aims to explore the effects of SUMO-1 gene silencing on GC cell proliferation and apoptosis. Methods: GC cells were cultured and divided into 5 groups: the blank group (without any transfection or treatment), the empty vector group (transfected with empty vector), the shRNA-SUMO-1-1 group (transfected with shRNA-SUMO-1-1 plasmid), the shRNASUMO- 1-2 group (transfected with shRNA-SUMO-1-2 plasmid), and the shRNA-SUMO-1-3 group (transfected with shRNA-SUMO-1-3 plasmid). Cell Counting Kit-8 (CCK-8) assay was performed to examine cell proliferation. Annexin V/PI staining combined with flow cytometry were used to detect cell apoptosis. Quantitative real-time polymerase chain reaction (qRTPCR) and Western blotting were employed to measure the mRNA and protein expressions of SUMO-1, P53, Bcl-2 and c-myc, respectively. Results: SUMO-1 mRNA and protein expressions were decreased after transfecting with shRNA-SUMO-1. Compared with the blank group, the shRNA-SUMO-1-1 group presented a remarkable decreased proliferation of SGC-7901 cells. Significant increase in cell apoptosis rate was observed. Bcl-2, c-myc and P53 expressions were declined after transfecting with shRNA-SUMO plasmid. Conclusion: Our study provided evidence that SUMO-1 gene silencing could decrease proliferation and promote apoptosis in GC cells. [ABSTRACT FROM AUTHOR]

Background: Small ubiquitin-like modifiers (SUMOs) conjugate to proteins post-translationally, thereby affecting target localization, activity and stability. Functional SUMO family members identified in the human placenta include SUMO-1 to SUMO-3, which are elevated in pre-eclampsia. Whether the fourth isoform, SUMO-4, plays a role in placental development and function remains unknown. Objectives: We tested the hypothesis that SUMO-4 is expressed in the human placenta and demonstrates altered SUMOylation in pre-eclamptic pregnancies. Methods: SUMO-4 mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) were measured in Jar cells, BeWo cells, first trimester placental villous explants and placental tissues across normal gestation and in pre-eclampsia. SUMO-4 expression in response to oxidative stress (H2O2: 0, 0.1, 1 and 5mM), as well as, hypoxia-reperfusion (O2: 1%, 8% and 20%) was measured. Lastly, SUMO-4 binding (covalently vs. non-covalently) to target proteins was investigated. Results: SUMO-4 mRNA and protein were unchanged across gestation. SUMO-4 was present in the villous trophoblast layer throughout gestation. SUMO-4 mRNA expression and protein levels were increased ~2.2-fold and ~1.8-fold in pre-eclamptic placentas compared to age-matched controls, respectively (p<0.01). SUMO-4 mRNA and protein expression increased in Jars, BeWos and first trimester placental explants with 5mM H2O2 treatment, as well as with exposure to hypoxia-reperfusion. SUMO-1 to SUMO-3 did not show consistent trends across models. SUMO-4 hyper-SUMOylation was predominantly covalent in nature. Conclusions: SUMO-4 is expressed in normal placental development. SUMO-4 expression was increased in pre-eclamptic placentas and in models of oxidative stress and hypoxic injury. These data suggests that SUMO-4 hyper-SUMOylation may be a potential post-translational mechanism in the stressed pre-eclamptic placenta. [ABSTRACT FROM AUTHOR]

The conjugation of small ubiquitin-like modifier (SUMO) to the target protein, namely, SUMOylation, is involved in the regulation of many important biological events including host-pathogen interaction. Some viruses have evolved to exploit the host SUMOylation machinery to modify their own protein. Retroviral Gag protein plays critical roles in the viral life cycle. The HIV-1 p6 and the Moloney murine leukemia virus CA have been reported to be conjugated with SUMO. In this study, we report for the first time, to our knowledge, the covalent conjugation of equine infectious anemia virus (EIAV) Gag with SUMO. The C -terminal p9 domain of Gag is a main target for SUMOylation and SUMO is attached to multiple sites of p9, including K30 whose mutation abolished p9 SUMOylation completely. The SUMOylation of p9, but not the p9-K30 mutant, was also detected in equine fibroblastic cells ATCC ® CCL-57™. Ubc9 and its C93 residue are indispensable for the SUMOylation of p9. Using confocal microscopy, it is found that EIAV Gag localizes primarily, if not exclusively, in the cytoplasm of the cell and the co-localization of EIAV Gag with Ubc9 was observed. Our findings that EIAV Gag is SUMOylated at p9-K30, together with previous findings on the defects of p9-K30 mutant in viral DNA translocation from cytoplasm to the nucleus, suggests that SUMOylation of Gag may be involved in such functions. [ABSTRACT FROM AUTHOR]

Pediatric high-grade gliomas represent 8–12% of all primary tumors of the nervous system in children. Five-year survival for these pediatric aggressive tumors is poor (15–35%) indicating the need to develop better treatments for pediatric high-grade gliomas. In this work we used SF188 and SJ-GBM2 cell lines to study the function of the ubiquitin carboxyl-terminal esterase L1 (UCHL1), a deubiquitinase de-regulated in several cancers, in pediatric high-grade gliomas. UCHL1 depletion in SF188 and SJ-GBM2 glioma cells was associated with decreased cell proliferation and invasion, along with a reduced ability to grow in soft agar and to form spheres (i.e. self-renewal measure). A 70% reduction in Wnt signaling was also observed in the SF188 and SJ-GBM2 UCHL1 knockdowns (KDs) using a TCF-dependent TOPflash reporter assay. Transcriptome comparisons of UCHL1 KDs versus vector control identified a list of 306 differentially expressed genes (at least 2-fold change; p <0.05) which included genes known to be involved in cancer like ACTA2, POSTN, LIF, FBXL7, FBXW11, GDF15, HEY2, but also potential novel genes such us IGLL5, ABCA4, AQP3, AQP4, CALB1, and ALK. Bioinformatics gene ontology (GO) analysis of these 306 genes revealed significant enrichment in “signal peptides”, “extracellular matrix”and “secreted proteins” GO Terms. “Angiogenesis and blood vessel development”, “neuron differentiation/development”, cell adhesion”, and “cell migration” also showed significant enrichment in our GO analysis. Top canonical pathways identified by Ingenuity Pathway Analysis (IPA) included “Clathrin-mediated Endocytosis Signaling” (p = 5.14x10-4), “Virus Entry via Endocytic Pathways” (p = 6.15x 10−4), and “High Mobility Group-Box 1 (HMGB1) Signaling” (p = 6.15x10-4). While FGF2, IL1B, TNF and PDGFB were predicted as top upstream regulators (p < 2x10-16) of the UCHL1 KD-associated transcriptome. Aberrant expression of UCHL1 in pediatric high-grade gliomas may promote cell invasion, transformation, and self-renewal properties, at least in part, by modulating Wnt/Beta catenin activity. UCHL1 might act as an oncogene in glioma within the gene network that imparts stem-like characteristics to these cancer cells. [ABSTRACT FROM AUTHOR]

Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle. [ABSTRACT FROM AUTHOR]

Background: Despite recent studies on the role of ubiquitin-related SUMO modifier in cell fate decisions, our understanding on precise molecular mechanisms of these processes is limited. Previously, we established that the SUMO isopeptidase SENP3 regulates chromatin assembly of the MLL1/2 histone methyltransferase complex at distinct HOX genes, including the osteogenic master regulator DLX3. A comprehensive mechanism that regulates SENP3 transcriptional function was not understood. Results: Here, we identified flightless-I homolog (FLII), a member of the gelsolin family of actin-remodeling proteins, as a novel regulator of SENP3. We demonstrate that FLII is associated with SENP3 and the MLL1/2 complex. We further show that FLII determines SENP3 recruitment and MLL1/2 complex assembly on the DLX3 gene. Consequently, FLII is indispensible for H3K4 methylation and proper loading of active RNA polymerase II at this gene locus. Most importantly, FLII-mediated SENP3 regulation governs osteogenic differentiation of human mesenchymal stem cells. Conclusion: Altogether, these data reveal a crucial functional interconnection of FLII with the sumoylation machinery that converges on epigenetic regulation and cell fate determination. [ABSTRACT FROM AUTHOR]

Arsenic exposure increases risk for cancers and is teratogenic in animal models. Here we demonstrate that small ubiquitin-like modifier (SUMO)- and folate-dependent nuclear de novo thymidylate (dTMP) biosynthesis is a sensitive target of arsenic trioxide (As2O3), leading to uracil misincorporation into DNA and genome instability. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and serine hydroxymethyltransferase (SHMT) generate 5,10-methylenetetrahydrofolate for de novo dTMP biosynthesis and translocate to the nucleus during S-phase, where they form a multienzyme complex with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR), as well as the components of the DNA replication machinery. As2O3 exposure increased MTHFD1 SUMOylation in cultured cells and in in vitro SUMOylation reactions, and increased MTHFD1 ubiquitination andMTHFD1 and SHMT1 degradation. As2O3 inhibited de novo dTMP biosynthesis in a dose-dependent manner, increased uracil levels in nuclear DNA, and increased genome instability. These results demonstrate that MTHFD1 and SHMT1, which are key enzymes providing one-carbon units for dTMP biosynthesis in the form of 5,10-methylenetetrahydrofolate, are direct targets of As2O3-induced proteolytic degradation, providing a mechanism for arsenic in the etiology of cancer and developmental anomalies. [ABSTRACT FROM AUTHOR]

The regulation of protein fate by modification with the small ubiquitin-related modifier (SUMO) plays an essential and crucial role in most cellular pathways. Sumoylation is highly dynamic due to the opposing activities of SUMO conjugation and SUMO deconjugation. SUMO conjugation is performed by the hierarchical action of E1, E2 and E3 enzymes, while its deconjugation involves SUMO-specific proteases. In this review, we summarize and compare the mechanistic principles of how SUMO gets conjugated to its substrate. We focus on the interplay of the E1, E2 and E3 enzymes and discuss how specificity could be achieved given the limited number of conjugating enzymes and the thousands of substrates. [ABSTRACT FROM AUTHOR]

Protein mechanics is a key component of many cellular and sub-cellular processes. The current review focuses on recent studies from our laboratory that probe the effect of sequence on the mechanical stability of structurally similar proteins and the unfolding mechanisms of multi-domain periplasmic binding proteins. Ubiquitin and small ubiquitin-related modifiers (SUMOs) are structurally similar and possess different mechanical stabilities, ubiquitin being stronger than SUMOs as revealed from their unfolding forces. These differences are plausibly due to the variation in number of inter-residue contacts. The unfolding potential widths determined from the pulling speed-dependent studies revealed that SUMOs are mechanically more flexible than ubiquitin. This flexibility of SUMOs plays a role in ligand binding and our single-molecule studies on SUMO interaction with SUMO binding motifs (SBMs) have shown that ligand binding decreases the SUMO flexibility and increases its mechanical stability. Studies on multi-domain periplasmic binding proteins have revealed that the unfolding energy landscape of these proteins is complex and they follow kinetic partitioning between two-state and multiple three-state pathways. [ABSTRACT FROM AUTHOR]

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express. [ABSTRACT FROM AUTHOR]

[Display omitted] • PDB Manipulator can detect and model many nonstandard amino acids and nucleic acids. • PDB Manipulator can detect and model ubiquitin/SUMO chains for a protein structure. • Various PTM modifications of Lys and Arg are supported in PDB Manipulator. • Users can edit the ligand structure for ligand parameterization using Marvin JS. Molecular modeling and simulation play important roles in biomedical research as they provide molecular-level insight into the underlying mechanisms of biological functions that are difficult to elucidate only with experiments. CHARMM-GUI (https://charmm-gui.org) is a web-based cyberinfrastructure that is widely used to generate various molecular simulation system and input files and thus facilitates and standardizes the usage of common and advanced simulation techniques. In particular, PDB Manipulator provides various chemical modification options as the starting point for most input generation modules in CHARMM-GUI. Here, we discuss recent additions to PDB Manipulator , such as non-standard amino acids/RNA substitutions, ubiquitylation and SUMOylation, Lys/Arg post-translational modifications, lipidation, peptide stapling, and improved parameterization options of small molecules. These additional features are expected to make complex PDB modifications easy for biomolecular modeling and simulation. [ABSTRACT FROM AUTHOR]