Podcasts about sumoylation

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550331v1?rss=1 Authors: Yucel, B. P., Al Momany, E., Evans, A. J., Seager, R., Wilkinson, K., Henley, J. M. Abstract: Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.16.549195v1?rss=1 Authors: Loya-Lopez, S. I., Allen, H. N., Duran, P., Calderon-Rivera, A., Gomez, K., Kumar, U., Shields, R., Zeng, R., Dwivedi, A., Saurabh, S., Korczeniewska, O. A., Khanna, R. Abstract: Dysregulation of voltage-gated sodium NaV1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/NaV1.7 interaction and increased functional activity of NaV1.7. Targeting this feed-forward regulation, we developed compound 194, which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of NaV1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we employed a comprehensive array of investigative approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of NaV1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a, Dpysl2, and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and NaV1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/NaV1.7 interaction, impeded NaV1.7 diffusion on the plasma membrane, and subsequently diminished NaV1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve (CCI-ION), 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating NaV1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Hugues de ThéCollège de France - Année 2022-2023Oncologie cellulaire et moléculaireColloque - Revisited Chemotherapy : SUMOylation Controls Acute Myeloid Leukemias Response to (Chemo)therapiesGuillaume Bossis, IGMM, CNRS, Montpellier, France

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539603v1?rss=1 Authors: Seager, R., Shree Ramesh, N., Cross, S., Guo, C., Wilkinson, K., Henley, J. Abstract: Mitochondrial fission regulates mitochondrial morphology, function, mitophagy and apoptosis. Fission is mediated by the GTPase dynamin related protein-1 (DRP1) and its recruitment to the outer mitochondrial membrane by DRP1 receptors. Mitochondrial fission factor (MFF) is considered the major pro-fission receptor, whereas the mitochondrial dynamics proteins (MiD49/51) sequester inactive DRP1 and facilitate the MFF-DRP1 interaction by forming a trimeric DRP1-MiD-MFF complex. Here, we identify MFF as a target of poly-SUMOylation at a single residue (Lys151). Following bioenergetic stress, AMPK phosphorylates MFF to promote its SUMOylation, a critical step in stress-induced fragmentation. MFF SUMOylation is not required for DRP1 recruitment from the cytosol but causes a rearrangement of the trimeric fission complex to displace MiD proteins. This alleviates MiD inhibition of DRP1 to facilitate formation of a fission-competent complex. Thus, our data demonstrate that MFF SUMOylation fine-tunes the ratio of MiD to DRP1 for the dynamic control of stress-induced mitochondrial fragmentation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538205v1?rss=1 Authors: Hirano, S., Udagawa, O., Kato-Udagawa, A. Abstract: Promyelocytic leukemia-nuclear bodies (PML-NBs) are dot-like protein assemblies and implicated in the pathogenesis of leukemia and viral infection. PML is the scaffold protein of PML-NB and its client proteins such as SUMO, DAXX, and Sp100 reside in PML-NBs. It is known that a short exposure to trivalent arsenic (As3+) induces the solubility change and the subsequent SUMOylation of PML, and the SUMO interacting motif (SIM) is not necessary for these biochemical changes. However, it has not been well studied how As3+ initiates or enhances the association of SUMO with PML and the other PML-NB client proteins. Here, we report that As3+ enhanced non-covalent association of PML with SUMO via the SUMO-SIM interaction which is dispensable for the solubility change and SUMOylation of PML. We also report that the As3+-induced solubility change of PML was not affected by ML792, a SUMO E1 enzyme inhibitor, even though the nuclear localization of SUMO2/3 and protein SUMOylation were halted by ML792. As3+ did not change the solubility of DAXX and SUMOylation enzymes such as SAE1, UBA2, and UBC9. In contrast, As3+ induced SUMOylation of Sp100 with a concomitant loss of its solubility like PML in human leukemia cell lines. Our current results indicate that both covalent and non-covalent associations of SUMO with PML are increased in As3+-exposed cells, and Sp100 may play a role in the maintenance of PML-NBs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.16.537086v1?rss=1 Authors: Zhang, J., Qiu, R., Bieger, B. D., Oakley, E., Oakley, B. R., Egan, M. J., Xiang, X. Abstract: Functions of protein SUMOylation remain incompletely understood in different cell types. The budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, but dynein-pathway components were not identified as SUMO-targets in the filamentous fungus Aspergillus nidulans. Via A. nidulans forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO-activation enzyme UbaB. Colonies of the ubaBQ247*, {triangleup}ubaB and {triangleup}sumO mutants looked similar and less healthy than the wild-type colony. In these mutants, about 10% of nuclei are connected by abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. Nuclei connected by chromatin bridges are mostly in interphase, suggesting that these bridges do not prevent cell-cycle progression. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO-targets being nuclear proteins, for example, topoisomerase II whose SUMOylation defect gives rise to chromatin bridges in mammalian cells. Unlike in mammalian cells, however, loss of SUMOylation in A. nidulans does not apparently affect the metaphase-to-anaphase transition, further highlighting differences in the requirements of SUMOylation in different cell types. Finally, loss of UbaB or SumO does not affect dynein- and LIS1-mediated early-endosome transport, indicating that SUMOylation is unnecessary for dynein or LIS1 function in A. nidulans. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.06.527258v1?rss=1 Authors: He, J., Guo, Y., Deng, R., Li, L., Huang, C., Chen, R., Wang, Y., Huang, J., Cheng, J., Chen, G.-Q., Zheng, J., Zhao, X., Yu, J. Abstract: Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and cell survival. Nuclear PTEN plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses non-homologous end joining (NHEJ) repair by directly dephosphorylating 53BP1. During DNA damage responses (DDR), p14ARF was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DNA-break sites. This was because that SUMO1 conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of BRCA1, which has been located to the core of 53BP1 foci on the chromatin during S/G2 stage. Further, these chromatin-loaded PTEN directly and specifically dephosphorylated pT543 of 53BP1, resulting in the dissociation of 53BP1-complex, which facilitated DNA end resection and ongoing HR repair. The SUMOylation-deficient PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking PTEN SUMOylation pathway with either an SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides the new mechanistic understanding of PTEN in HR repair and clinical intervention of chemo-resistant tumors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.29.526122v1?rss=1 Authors: Johnson, Q., Hadjian, M., Bah, A., Smith, S., Kosa, E., Agbas, A. Abstract: The assignment of blood-based biomarkers for neurodegenerative diseases is of great clinical value. Well-developed and validated blood-based biomarkers can serve in early diagnosis and prognosis as well as aid in patient screening when recruiting for clinical trials. We attempted to establish a portfolio for post-translationally modified TAR DNA/RNA-binding protein (TDP-43), a regulator of nuclear transcription, in platelet cytosol obtained from patients with Alzheimer disease (AD) comparing to age-matched healthy subjects and a disease control cohort. We aimed to identify the most prominent post-translational modifications of TDP-43 as an AD-relevant biomarker and to demonstrate that such an assessment can be performed in peripheral blood. We have isolated TDP-43 protein from human platelet cytosol utilizing an Immunoaffinity chromatography. The eluates were immunoprobed with a series of antibodies raised against post-translationally modified proteins. We employed a capillary electrophoretic immunoassay (CEI) to assess the phosphorylated TDP-43 profile. We observed that SUMOylation, phosphorylation, ubiquitination, and cysteine oxidation of TDP-43 are more prominent in platelet cytosol of AD patients as compared to control subjects. These studies will pave the way for identifying disease-specific TDP-43 derivatives that can be potential biomarkers for early diagnosis and the development of therapeutics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.08.523158v1?rss=1 Authors: Mueller, J., Furlan, M., Settele, D., Grupp, B., Johnsson, N. Abstract: Ubiquitylation and phosphorylation control composition and architecture of the cell separation machinery in yeast and other eukaryotes. The significance of septin sumoylation on cell separation remained an enigma. Septins form an hourglass structure at the bud neck of yeast cells that transforms into a split septin double ring during mitosis. We discovered that sumoylated septins recruit the cytokinesis checkpoint protein Fir1 to the peripheral side of the septin hourglass. Subsequent de-sumoylation and synchronized binding to the scaffold Spa2 relocate Fir1 in a seamless transition between the split septin rings. Fir1 binds and carries Skt5 on its route to the division plane where the Fir1-Skt5 complex serves as receptor for chitin synthase III. We propose that the opposite positioning of the sumoylated septins and Spa2 creates a tension across the ring that upon de-sumoylation tunnels the membrane-bound Fir1-Skt5 complex through a transiently permeable septin diffusion barrier. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.11.516192v1?rss=1 Authors: Orsini, F., Argyrousi, E. K., Restelli, E., Ford, L. K., Takamura, H., Matsuzaki, S., Zentilin, L., Pascente, R., Kanaan, N. M., Soni, R., Katayama, T., Chiesa, R., Forloni, G., Kosik, K. S., Kandel, E. R., Fraser, P. E., Arancio, O., Fioriti, L. Abstract: Abnormal intracellular accumulation of Tau aggregates is a hallmark of Alzheimer's disease (AD) and other Tauopathies, such as Frontotemporal dementia (FTD), which can be caused by mutations of Tau. Mutated and pathological Tau can undergo a range of post-translational modifications (PTMs) that might trigger or modulate disease pathology. Recent studies indicate that modification of wild type Tau by Small ubiquitin-like modifier SUMO isoform 1 (SUMO1) controls Tau hyperphosphorylation and aggregation, suggesting that SUMOylation acts as a central regulator of Tau's biochemical properties. Besides SUMO1, Tau is modified by SUMO2/3, however the consequences of this modification have not been investigated. Here, using viral approaches on primary hippocampal neurons, transgenic mice expressing mutant Tau and SUMO2, and iPSC-derived neurons from FTD patients, we evaluated whether SUMO2/3 conjugation modifies the neurodegenerative disease pathology associated with the aggregation-prone mutant Tau P301L, P301S, and R406W variants. We found that mutant forms of Tau are targets of SUMO2/3, and SUMO2/3 conjugation is neuroprotective. Importantly, expression of mutant Tau is accompanied by a significant reduction of SUMO2/3 conjugation levels, and restoring levels of SUMO2 reduces mutant Tau aggregation and phosphorylation in all model systems Furthermore, overexpression of SUMO2 restores levels of pre- and post-synaptic markers, associated with a complete rescue of the LTP and memory deficits in transgenic mice expressing mutant Tau. These findings bring to light the potential therapeutic implication of manipulating SUMO conjugation to detoxify Tau through PTM-based approaches. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.02.514898v1?rss=1 Authors: Strachan, J., Leidecker, O., Spanos, C., Le Coz, C., Chapman, E., Arsenijevic, A., Zhang, H., Zhao, N., Bayne, E. H. Abstract: Regulation by the small modifier SUMO is heavily dependent on spatial control of enzymes that mediate the attachment and removal of SUMO on substrate proteins. Here we show that in fission yeast, delocalisation of the SUMO protease Ulp1 from the nuclear envelope results in centromeric defects that can be attributed to hyper-SUMOylation at the nuclear periphery. Unexpectedly, we find that while this localised hyper-SUMOylation impairs centromeric silencing, it can also enhance centromere clustering. Moreover, both effects are at least partially dependent on SUMOylation of the inner nuclear membrane protein Lem2. Lem2 has previously been implicated in diverse biological processes including the promotion of both centromere clustering and silencing, but how these distinct activities are coordinated was unclear; our observations suggest a model whereby SUMOylation may serve as a regulatory switch, modulating Lem2 interactions with competing partner proteins to balance its roles in alternative pathways. Our findings also reveal a previously unappreciated role for SUMOylation in promoting centromere clustering. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.09.507035v1?rss=1 Authors: Suk, T. R., Nguyen, T. T., Fisk, Z. A., Mitkovski, M., Geertsma, H. M., Parmasad, J.-L. A., Heer, M. M., Callaghan, S. M., Brose, N., Tirard, M., Rousseaux, M. W. C. Abstract: SUMOylation is an evolutionarily conserved and essential mechanism whereby Small Ubiquitin Like Modifiers, or SUMO proteins (Sumo in mice), are covalently bound to protein substrates in a highly dynamic and reversible manner. SUMOylation is involved in a variety of basic neurological processes including learning and memory, and central nervous system development, but is also linked with neurological disorders. However, studying SUMOylation in vivo remains challenging due to limited tools to study Sumo proteins and their targets in their native context. More complexity arises from the fact that Sumo1 and Sumo2 are ~50% homologous, whereas Sumo2 and Sumo3 are nearly identical and indistinguishable with antibodies. While Sumo paralogues can compensate for one another's loss, Sumo2 is highest expressed and only paralog essential for embryonic development making it critical to uncover roles specific to Sumo2 in vivo. To further examine the roles of Sumo2, and to begin to tease apart the redundancy and similarity between key Sumo paralogs, we generated (His6-)HA epitope-tagged Sumo2 knock-in mouse alleles, expanding the current Sumo knock-in mouse tool-kit comprising of the previously generated His6-HA-Sumo1 knock-in model. Using these HA-Sumo mouse lines, we performed whole brain imaging and mapping to the Allen Brain Atlas to analyze the relative distribution of the Sumo1 and Sumo2 paralogues in the adult mouse brain. We observed differential staining patterns between Sumo1 and Sumo2, including a partial localization of Sumo2 in nerve cell synapses of the hippocampus. Combining immunoprecipitation with mass spectrometry, we identified native substrates targeted by Sumo1 or Sumo2 in the mouse brain. We validated select hits using proximity ligation assays, further providing insight into the subcellular distribution of neuronal Sumo2-conjugates. These mouse models thus serve as valuable tools to study the cellular and biochemical roles of SUMOylation in the central nervous system. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Karuppiah Kannan, Senior Director - Global Program Leader at Takeda Pharmaceuticals, discusses early results of a phase 1/2 study evaluating subasumstat (TAK-981) in combination with rituximab in multiple subsets of CD20-positive relapsed/refractory non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL) and marginal zone lymphoma (MZL). The results of this study were recently presented at The American Society of Hematology Meeting & Exposition (ASH 2021).Subasumstat is an investigational, first-in-class small-molecule inhibitor of SUMOylation, which mediates cell cycle progression. In preclinical trials, subasumstat was shown to add synergistic benefit when combined with rituximab in non-Hodgkin lymphoma models.

For more details, visit #DrGPCR​​​ Podcast Episode #56 page: http://www.drgpcr.com/episode-56-with-dr-adriano-marchese/ ------------------------------------------- Adriano Marchese is a Professor of Biochemistry at the Medical College of Wisconsin. Adriano received his Bachelor of Science degree in Pharmacology in 1991 from the University of Toronto. He continued his graduate studies at the University of Toronto where he earned his MSc (1994) and Ph.D. (1998) in Pharmacology. He then moved to Thomas Jefferson University in Philadelphia, PA, for his postdoctoral training in Jeff Benovic's laboratory studying the regulation of G protein-coupled receptor trafficking and signaling. In 2004 Adriano joined the faculty of the Department of Pharmacology at Loyola University Chicago. In 2016 he decided to move his lab to the Medical College of Wisconsin in Milwaukee, WI. Adriano's research has contributed to our understanding of the role that ubiquitin plays in GPCR signaling and trafficking. His laboratory is interested in understanding the mechanisms that govern spatial and temporal regulation of GPCR signaling by -arrestins and post-translational modifications (PTMs), such as phosphorylation, ubiquitination, and SUMOylation. His lab has shown a role for -arrestins and PTMs in GPCR trafficking and signaling and has leveraged this knowledge to reveal the spatial and temporal requirements for GPCR activation of signaling pathways related to cell survival, proliferation, and migration. The ultimate goal of Adriano's research is to target novel aspects of GPCR signaling for therapeutic development. ------------------------------------------- Imagine a world in which the vast majority of us are healthy. The #DrGPCR Ecosystem is all about dynamic interactions between us who are working towards exploiting the druggability of #GPCR's. We aspire to provide opportunities to connect, share, form trusting partnerships, grow, and thrive together. To build our #GPCR Ecosystem, we created various enabling outlets. For more details, visit our website http://www.DrGPCR.com/Ecosystem/ ------------------------------------------- Are you a #GPCR professional? - Register to become a Virtual Cafe speaker http://www.drgpcr.com/virtual-cafe/ - Subscribe to our Monthly Newsletter http://www.drgpcr.com/newsletter/ - Listen and subscribe to #DrGPCR Podcasts http://www.drgpcr.com/podcast/ - Support #DrGPCR Ecosystem with your Donation. http://www.drgpcr.com/sponsors/ - Reserve your spots for the next #DrGPCR Virtual Cafe http://www.drgpcr.com/virtual-cafe/

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.05.327064v1?rss=1 Authors: Kmiecik, S. W., Drzewicka, K., Melchior, F., Mayer, M. P. Abstract: The heat shock response (HSR) is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Posttranslational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by SUMO (small ubiquitin-like modifier) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low level SUMOylation at other sites. None of the tested E3 SUMO ligases increased SUMOylation efficacy as compared to the level in the presence of high concentrations of the E2 Ubc9. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA-bound, and SUMOylation of Hsf1 does neither alter DNA binding affinity nor does it affect Hsc70 and DnaJB1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the HSR. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.05.326249v1?rss=1 Authors: Mas, A., Castano-Miquel, L., Carretero-Paulet, L., Colome, N., Canals, F., Lois, L. M. Abstract: Post-translational modification by Small Ubiquitin-related Modifier (SUMO) is an essential regulatory mechanism in eukaryotes. In the cell, SUMO conjugates are highly enriched in the nucleus and, consistently, SUMOylation machinery components are mainly nuclear. Nonetheless, cytosolic SUMO targets also exist and the mechanisms that facilitate SUMO conjugation in the cytosol are unknown. Here, we show that the nuclear localization of the Arabidopsis SUMO activating enzyme large subunit SAE2 is dependent on two nuclear localization signals, the canonical NLS1 and the non-canonical NLS2 identified and validated here. NLS2 is proteolytic processed from SAE2 during seed development, facilitating SAE2 enrichment in the cytosol. Results obtained using transgenic plants expressing different SAE2 proteoforms suggest that SAE2 cytosolic enrichment could constitute a rapid signal for growth arrest. Phylogenetic studies indicated that the Arabidopsis NLS1-NLS2 structural organization is conserved only in seed plants, providing a potential evolutionary role of cytosolic SUMOylation in seed appearance. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.318774v1?rss=1 Authors: Gomez, K., Ran, D., Madura, C. L., Moutal, A., Khanna, R. Abstract: Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). If not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male and female CRMP2K374A/K374A mice, increased pain sensitivity in allodynia-resistant animals. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non SUMOylated CRMP2-mediated internalization of sodium channels in vivo. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.15.299115v1?rss=1 Authors: Peeples, W., Rosen, M. K. Abstract: Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymatic reaction rates through increased concentrations of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liquid-liquid phase separation of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase separation threshold lead to a quantitative model in which reactions in condensates are accelerated by mass action and by changes in substrate KM, likely due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and by physically organizing them. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.03.281881v1?rss=1 Authors: Park, S. L. L., Ramirez Jarquin, U. N., Shahani, N., Rivera, O., Sharma, M., McManus, F. P., Thibault, P., Subramaniam, S. Abstract: The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications, however, underlying mechanisms remains unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, G{beta}L, is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that G{beta}L is SUMOylated at lysine sites K86, K215, K245, K261 and K305. We found that SUMO depletion reduces mTOR-Raptor and mTOR-Rictor complex formation and diminishes nutrient-induced mTOR signaling. Furthermore, we found that reconstitution with WT G{beta}L but not SUMOylation defective KR mutant G{beta}L promote mTOR signaling in G{beta}L-depleted cells. Taken together, we report for the very first time that SUMO modifies G{beta}L, influences the assembly of mTOR protein complexes, and regulates mTOR activity. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.16.100032v1?rss=1 Authors: Ford, L., Asok, A., Tripp, A. D., Parro, C., Fitzpatrick, M., de Solis, C. A., Shafiian, N., Fioriti, L., Soni, R. K., Kandel, E. Abstract: Biomolecular condensates, membraneless organelles found throughout the cell, play critical roles in many aspects of cellular function. Ribonucleoprotein granules (RNPs), a type of biomolecular condensate found in neurons that are necessary for local protein synthesis and are involved in long-term potentiation (LTP). Several RNA-binding proteins present in RNPs are necessary for the synaptic plasticity involved in LTP and long-term memory. Most of these proteins possess low complexity motifs, allowing for increased promiscuity. We explore the role the low complexity motif plays for RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CPEB3), a protein necessary for long-term memory persistence. We found that RNA binding and SUMOylation are necessary for CPEB3 localization to the P body, thereby having functional implications on translation. Here, we investigate the role of the low complexity motif of CPEB3 and find that it is necessary for P body localization and downstream targeting for local protein synthesis. Copy rights belong to original authors. Visit the link for more info

Paul FRASER, Jeno Diener Chair in Neurodegenerative Diseases, Tanz Centre and Department of Medical Biophysics, University of Toronto, CANADA speaks on "SUMOylation pathways in neuronal function and neurodegeneration". This movie has been recorded at ICGRB Trieste, Italy.

The SUMO (small ubiquitin-related modified protein) family of proteins have been linked to cell cycle progression since their discovery. Bio-Rad’s top tips for studying this transient post translational modification feature in our podcast.

Guy Salvesen explains how arsenic trioxide treatment switches which SUMO variant is conjugated to an oncoprotein, thus triggering its degradation.

Posttranslational modifications (PTMs) of proteins by covalent attachment of functional groups (like phosphorylation, acetylation, methylation, glycosylation, etc.) are of key importance for the cell as they regulate various aspects of protein behavior after its synthesis, e.g., dictate protein interaction properties, change catalytic activity of enzymes, induce conformational changes, guide subcellular localization and determine protein stability. A special class of protein PTMs is the conjugation of small proteins of the ubiquitin family to typically acceptor lysine residues of the substrates. The reversible nature of this PTM and the presence of dedicated domains that specifically recognize modified substrates make this type of protein modification instrumental for the regulation of numerous biological pathways. For ubiquitylation, strong substrate selectivity due to the presence of highly diversified conjugation machinery is characteristic and well studied, especially in case of ubiquitin’s proteolytic role. On the contrary, much less is known about the principles of substrate specificity and mechanisms of PTM action in the ubiquitin-like protein SUMO modification system. Despite the fact that SUMOylation specifically targets hundreds of substrates and major conjugation steps are identical with ubiquitin system, strikingly only a handful of enzymes operate in the SUMO pathway, suggesting that other principles of substrate selectivity must apply and perhaps distinct mechanisms of PTM action exist in the SUMO pathway. Moreover, the recognition of SUMO modification is surprisingly simple and relies mainly on a short hydrophobic sequence known as SUMO-interacting motif (SIM), in striking contrast to the ubiquitin system, where numerous ubiquitin-binding domains exist with different interaction specificities. All these, together with the observations that SUMO conjugation machinery seems rather promiscuous in vitro, that typically only a small fraction of a protein is being SUMOylated at a given time, and that specific SUMOylation-defective mutants often exhibit no obvious phenotypes, whereas SUMO pathway mutants do, emphasize the question of substrate specificity in the SUMO system and suggest other principles of SUMO action on its substrates. Here, we address the question of SUMOylation specificity and function using DNA double-strand break (DSB) repair pathway via homologous recombination (HR) as a case study because of its strong ties to the SUMO system. First, using SILAC-based proteomic approach we show that proteins acting in the same DNA repair pathway become collectively SUMOylated upon a specific stimulus (HR factors – upon DSB induction; nucleotide excision repair factors – upon exposure to UV light), suggesting that SUMO machinery often targets protein groups within the same pathway. Then, focusing on the DSB repair we find that DNA-bound SUMO ligase Siz2 catalyzes collective multisite SUMOylation of a whole set of HR factors. Repair proteins are loaded onto resected single-stranded DNA (ssDNA) in the vicinity of the ligase, thus making exposure of ssDNA a precise trigger for modification. Protein group SUMOylation fosters physical interactions between the HR proteins engaged in DNA repair, because not only that they become collectively modified at multiple SUMO-acceptor sites, but they also possess multiple SIMs, which promote SUMO-SIM mediated complex formation. Only wholesale elimination of SUMOylation of the core HR proteins significantly affects the HR pathway by slowing down DNA repair, suggesting that SUMO acts synergistically on several proteins. Thus, we show that SUMOylation collectively targets functionally engaged protein group rather than individual proteins, whereas localization of modification enzymes and specific triggers ensure substrate specificity.

Thu, 2 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12361/ https://edoc.ub.uni-muenchen.de/12361/2/Shan_Bing.pdf Shan, Bing ddc:570, ddc:500, Fakultät f

A novel high-throughput screening approach that combines yeast mutant arrays with high-content imaging provides fresh insights into mitotic spindle disassembly. This biosights episode presents the paper by Vizeacoumar et al. from the January 11, 2010 issue of the Journal of Cell Biology, and includes interviews with authors Brenda Andrews and Charles Boone, and JCB editorial board member John Aitchison. Produced by Eun Choi.   Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Small ubiquitin-related modifier (SUMO) is a protein that is attached to lysine residues in a variety of target proteins. Sumoylation of proteins can alter their intracellular localisation, stability, activity and interaction with other proteins. The pathway of sumoylation is analogous to that of ubiquitination. The reaction is ATP dependent and requires the E1-activating enzyme (Aos1/Uba2), the E2-conjugation enzyme (Ubc9) and for most target proteins SUMO E3 ligases. The aim of this study was to characterise the SUMO E1 enzyme, a heterodimer consisting of the subunits Aos1 and Uba2. On one hand I characterised an Uba2 splice variant, which lacks one exon encoding 50 amino acids. Using RT-PCR I could determine the tissue specific distribution of the Uba2 splice variant. I furthermore showed that Uba2 variant protein is still able to form an active E1 enzyme complex with Aos1. I could demonstrate that variant Aos1/Uba2 complex is fully active in RanGAP1 sumoylation with SUMO1 or SUMO2. This finding was surprising in light of the missing amino acids, and will have implications for the understanding of E1 function. A large part of my work was dedicated to the identification and characterization of a novel SUMO substrate called ELKS. According to literature, ELKS proteins have been linked to intracellular membrane traffic and NFB signaling pathways. I identified ELKS in membrane fractions as a binding partner for the Aos1 subunit of the SUMO E1 enzyme and confirmed in vivo interaction with ELKS antibodies that I generated. Because recombinant proteins did not interact directly, I searched for potential bridging factors. Neither SUMO nor Ubc9 or Rab6 (one ELKS partner) mediated interaction between ELKS and Aos1. Performing a large scale immunoprecipitation and analysis by mass spectrometry, I could find several candidates, including nucleoporin RanBP2, a SUMO E3 ligase. This suggested that ELKS may be a target for sumoylation. Indeed, I could show that ELKS was SUMO-modified in vivo and in vitro. Moreover, RanBP2 enhanced ELKS sumoylation. By mass spectrometry I identified two SUMO acceptor sites in ELKS. Mutation of these two residues had no effect on ELKS localisation, but strongly inhibited ELKS induced NFB activation. In conclusion, work described in this thesis implicates sumoylation as an important mechanism for ELKS function in NFkB signaling.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.20.049106v1?rss=1 Authors: Moutal, A., Cai, S., Yu, J., Stratton, H. J., Chefdeville, A., Gomez, K., Ran, D., Madura, C. L., Boinon, L., Soto, M., Zhou, Y., Shan, Z., Chew, L. A., Rodgers, K. A., Khanna, R. Abstract: The sodium channel NaV1.7 is a master regulator of nociceptive neuronal firing. Mutations in this channel can result in painful conditions as well as produce insensitivity to pain. Despite being recognized as a 'poster child' for nociceptive signaling and human pain, targeting NaV1.7 has not yet produced a clinical drug. Recent work has illuminated the NaV1.7 interactome, offering insights into the regulation of these channels and identifying potentially new druggable targets. Amongst the regulators of NaV1.7 is the cytosolic collapsin response mediator protein 2 (CRMP2). CRMP2, modified at Lysine 374 (K374) by addition of a small ubiquitin-like modifier (SUMO), bound NaV1.7 to regulate its membrane localization and function. Corollary to this, preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in rats with neuropathic pain. Notably, loss of CRMP2 SUMOylation did not compromise other innate functions of CRMP2. To further elucidate the in vivo role of CRMP2 SUMOylation in pain, we generated CRMP2 K374A knock-in (CRMP2K374A/K374A) mice in which Lys374 was replaced with Ala. CRMP2K374A/K374A mice had reduced NaV1.7 membrane localization and function in female, but not male, sensory neurons. Behavioral appraisal of CRMP2K374A/K374A mice demonstrated no changes in depressive or repetitive, compulsive-like behaviors, and a decrease in noxious thermal sensitivity. No changes were observed in CRMP2K374A/K374A mice to inflammatory, acute, or visceral pain. In contrast, in a neuropathic model, CRMP2K374A/K374A mice failed to develop persistent mechanical allodynia. Our study suggests that CRMP2 SUMOylation-dependent control of peripheral NaV1.7 is a hallmark of chronic, but not physiological, neuropathic pain. Copy rights belong to original authors. Visit the link for more info