SUMO-conjugating Enzyme Ubc9 Research Articles

PIAS1 S510G variant acts as a genetic modifier of spinocerebellar ataxia type 3 by selectively impairing mutant ataxin-3 proteostasis

Dysregulated protein homeostasis, characterized by abnormal protein accumulation and aggregation, is a key contributor to the progression of neurodegenerative disorders such as Huntington's disease and spinocerebellar ataxia type 3 (SCA3). Previous studies have identified PIAS1 gene variants in patients with late-onset SCA3 and Huntington's disease. This study aims to elucidate the role of PIAS1 and its S510G variant in modulating the pathogenic mechanisms of SCA3. Through in vitro biochemical analyses and in vivo assays, we demonstrate that PIAS1 stabilizes both wild-type and mutant ataxin-3 (ATXN3). The PIAS1 S510G variant, however, selectively reduces the stability and SUMOylation of mutant ATXN3, thereby decreasing its aggregation and toxicity while maintaining the stability of wild-type ATXN3. This effect is mediated by a weakened interaction with the SUMO-conjugating enzyme UBC9 in the presence of mutant ATXN3. In Drosophila models, downregulation of dPIAS1 resulted in reduced levels of mutant ATXN3 and alleviated associated phenotypes, including retinal degeneration and motor dysfunction. Our findings suggest that the PIAS1 S510G variant acts as a genetic modifier of SCA3, highlighting the potential of targeting SUMOylation as a therapeutic strategy for this disease.

Protein SUMOylation promotes cAMP-independent EPAC1 activation

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

Repression of the SUMO-conjugating enzyme UBC9 is associated with lowered double minutes and reduced tumor progression

ABSTRACT Double minutes (DMs), extrachromosomal gene fragments found within certain tumors, have been noted to carry onco- and drug resistance genes contributing to tumor pathogenesis and progression. After screening for SUMO-related molecule expression within various tumor sample and cell line databases, we found that SUMO-conjugating enzyme UBC9 has been associated with genome instability and tumor cell DM counts, which was confirmed both in vitro and in vivo. Karyotyping determined DM counts post-UBC9 knockdown or SUMOylation inhibitor 2-D08, while RT-qPCR and Western blot were used to measure DM-carried gene expression in vitro. In vivo, fluorescence in situ hybridization (FISH) identified micronucleus (MN) expulsion. Western blot and immunofluorescence staining were then used to determine DNA damage extent, and a reporter plasmid system was constructed to detect changes in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Our research has shown that UBC9 inhibition is able to attenuate DM formation and lower DM-carried gene expression, in turn reducing tumor growth and malignant phenotype, via MN efflux of DMs and lowering NHEJ activity to increase DNA damage. These findings thus reveal a relationship between heightened UBC9 activity, increased DM counts, and tumor progression, providing a potential approach for targeted therapies, via UBC9 inhibition.

The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

Severe COVID-19 patients exhibit impaired IFN-I response due to decreased IFN-β production, allowing persistent viral load and exacerbated inflammation. While the SARS-CoV-2 nucleocapsid (N) protein has been implicated in inhibiting innate immunity by interfering with IFN-β signaling, the specific underlying mechanism still needs further investigation for a comprehensive understanding. This study reveals that the SARS-CoV-2 N protein enhances interaction between the human SUMO-conjugating enzyme UBC9 and MAVS. Increased MAVS-UBC9 interaction leads to enhanced SUMOylation of MAVS, inhibiting its ubiquitination, resulting in the inhibition of phosphorylation events involving IKKα, TBK1, and IRF3, thus disrupting IFN-β signaling. This study highlights the role of the N protein of SARS-CoV-2 in modulating the innate immune response by affecting the MAVS SUMOylation and ubiquitination processes, leading to inhibition of the IFN-β signaling pathway. These findings shed light on the complex mechanisms utilized by SARS-CoV-2 to manipulate the host's antiviral defenses and provide potential insights for developing targeted therapeutic strategies against severe COVID-19.

UBC9 stabilizes PFKFB3 to promote aerobic glycolysis and proliferation of glioblastoma cells

Cancer cells prefer to utilizing aerobic glycolysis to generate energy and anabolic metabolic intermediates for cell growth. However, whether the activities of glycolytic enzymes can be regulated by specific posttranslational modifications, such as SUMOylation, in response to oncogenic signallings, thereby promoting the Warburg effect, remain largely unclear. Here, we demonstrate that phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key glycolytic enzyme, interacts with SUMO-conjugating enzyme UBC9 and is SUMOylated at K302 in glioblastoma cells. Expression of UBC9, which competitively prevents the binding of ubiquitin E3 ligase APC/C to PFKFB3 and subsequent PFKFB3 polyubiquitination, increases PFKFB3 stability and expression. Importantly, EGFR activation increases the interaction between UBC9 and PFKFB3, leading to increased SUMOylation and expression of PFKFB3. This increase is blocked by inhibition of EGFR-induced AKT activation whereas expression of activate AKT by itself was sufficient to recapitulate EGF-induced effect. Knockout of PFKFB3 expression decreases EGF-enhanced lactate production and GBM cell proliferation and this decrease was fully rescued by reconstituted expression of WT PFKFB3 whereas PFKFB3 K302R mutant expression abrogates EGF- and UBC9-regulated lactate production and GBM cell proliferation. These findings reveal a previously unknown mechanism underlying the regulation of the Warburg effect through the EGFR activation-induced and UBC9-mediated SUMOylation and stabilization of PFKFB3.

A stress-induced cilium-to-PML-NB route drives senescence initiation

Cellular senescence contributes to tissue homeostasis and age-related pathologies. However, how senescence is initiated in stressed cells remains vague. Here, we discover that exposure to irradiation, oxidative or inflammatory stressors induces transient biogenesis of primary cilia, which are then used by stressed cells to communicate with the promyelocytic leukemia nuclear bodies (PML-NBs) to initiate senescence responses in human cells. Mechanistically, a ciliary ARL13B-ARL3 GTPase cascade negatively regulates the association of transition fiber protein FBF1 and SUMO-conjugating enzyme UBC9. Irreparable stresses downregulate the ciliary ARLs and release UBC9 to SUMOylate FBF1 at the ciliary base. SUMOylated FBF1 then translocates to PML-NBs to promote PML-NB biogenesis and PML-NB-dependent senescence initiation. Remarkably, Fbf1 ablation effectively subdues global senescence burden and prevents associated health decline in irradiation-treated mice. Collectively, our findings assign the primary cilium a key role in senescence induction in mammalian cells and, also, a promising target in future senotherapy strategies.

PACT promotes the metastasis of basal-like breast cancer through Rac1 SUMOylation and activation.

Most basal-like breast cancers (BLBCs) are triple-negative breast cancers (TNBCs), which is associated with high malignancy, high rate of recurrence and distant metastasis, and poor prognosis among all types of breast cancer. However, there are currently no effective therapies for BLBC. Furthermore, chemoresistance limits the therapeutic options for BLBC treatment. In this study, we screen out protein activator of the interferon-induced protein kinase (PACT) as an essential gene in BLBC metastasis. We find that high PACT expression level was associated with poor prognosis among BLBC patients. In vivo and in vitro investigations indicated that PACT could regulate BLBC metastasis by interacting with SUMO-conjugating enzyme Ubc9 to stimulate the SUMOylation and thus consequently the activation of Rac1. BLBC patients receiving chemotherapy presents poorer prognosis with PACT high expression, and PACT disruption sensitizes experimental mammary tumor metastases to chemotherapy, thus providing insights to consider PACT as a potential therapeutic target to overcome acquired chemoresistance in BLBC.

Cell specific regulation of NaV1.7 activity and trafficking in rat nodose ganglia neurons

The voltage-gated sodium NaV1.7 channel sets the threshold for electrogenesis. Mutations in the gene encoding human NaV1.7 (SCN9A) cause painful neuropathies or pain insensitivity. In dorsal root ganglion (DRG) neurons, activity and trafficking of NaV1.7 are regulated by the auxiliary collapsin response mediator protein 2 (CRMP2). Specifically, preventing addition of a small ubiquitin-like modifier (SUMO), by the E2 SUMO-conjugating enzyme Ubc9, at lysine-374 (K374) of CRMP2 reduces NaV1.7 channel trafficking and activity. We previously identified a small molecule, designated 194, that prevented CRMP2 SUMOylation by Ubc9 to reduce NaV1.7 surface expression and currents, leading to a reduction in spinal nociceptive transmission, and culminating in normalization of mechanical allodynia in models of neuropathic pain. In this study, we investigated whether NaV1.7 control via CRMP2-SUMOylation is conserved in nodose ganglion (NG) neurons. This study was motivated by our desire to develop 194 as a safe, non-opioid substitute for persistent pain, which led us to wonder how 194 would impact NaV1.7 in NG neurons, which are responsible for driving the cough reflex. We found functioning NaV1.7 channels in NG neurons; however, they were resistant to downregulation via either CRMP2 knockdown or pharmacological inhibition of CRMP2 SUMOylation by 194. CRMP2 SUMOylation and interaction with NaV1.7 was consered in NG neurons but the endocytic machinery was deficient in the endocytic adaptor protein Numb. Overexpression of Numb rescued CRMP2-dependent regulation on NaV1.7, rendering NG neurons sensitive to 194. Altogether, these data point at the existence of cell-specific mechanisms regulating NaV1.7 trafficking.

SUMOylation of microtubule-cleaving enzyme KATNA1 promotes microtubule severing and neurite outgrowth

Katanin p60 ATPase-containing subunit A1 (KATNA1) is a microtubule-cleaving enzyme that regulates the development of neural protrusions through cytoskeletal rearrangements. However, the mechanism underlying the linkage of the small ubiquitin-like modifier (SUMO) protein to KATNA1 and how this modification regulates the development of neural protrusions is unclear. Here we discovered, using mass spectrometry analysis, that SUMO-conjugating enzyme UBC9, an enzyme necessary for the SUMOylation process, was present in the KATNA1 interactome. Moreover, GST-pull down and co-immunoprecipitation assays confirmed that KATNA1 and SUMO interact. We further demonstrated using immunofluorescence experiments that KATNA1 and the SUMO2 isoform colocalized in hippocampal neurites. We also performed a bioinformatics analysis of KATNA1 protein sequences to identify three potentially conserved SUMOylation sites (K77, K157, and K330) among vertebrates. Mutation of K330, but not K77 or K157, abolished KATNA1-induced microtubule severing and decreased the level of binding observed for KATNA1 and SUMO2. Cotransfection of SUMO2 and wildtype KATNA1 in COS7 cells increased microtubule severing, whereas no effect was observed after cotransfection with the K330R KATNA1 mutant. Furthermore, in cultured hippocampal neurons, overexpression of wildtype KATNA1 significantly promoted neurite outgrowth, whereas the K330R mutant eliminated this effect. Taken together, our results demonstrate that the K330 site in KATNA1 is modified by SUMOylation and SUMOylation of KATNA1 promotes microtubule dynamics and hippocampal neurite outgrowth.

Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9.

Ubiquitylation—the attachment of ubiquitin (Ub) to proteins in eukaryotic cells—involves a vast number of enzymes from three different classes, resulting in heterogeneous attachment sites and ubiquitin chains. Recently, we introduced lysine acylation using conjugating enzymes (LACE) in which ubiquitin or peptide thioester is site-specifically transferred to a short peptide tag by the SUMO E2 conjugating enzyme Ubc9. This process, however, suffers from slow kinetics—due to a rate-limiting thioester loading step—and the requirement for thioesters restricts its use to in vitro reactions. To overcome these challenges, we devised a chimeric E1 containing the Ub fold domain of the SUMO E1 and the remaining domains of the Ub E1, which activates and loads native Ub onto Ubc9 and obviates the need for Ub thioester in LACE. The chimeric E1 was subjected to directed evolution to improve its apparent second-order rate constant (kcat/KM) 400-fold. We demonstrate the utility of the chimeric E1 by site-specific transfer of mono- and oligo-Ub to various target proteins in vitro. Additionally, the chimeric E1, Ubc9, Ub, and the target protein can be coexpressed in Escherichia coli for the facile preparation of monoubiquitylated proteins.

Small molecule targeting NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces and prevents pain chronification in a mouse model of oxaliplatin-induced neuropathic pain

Treatment with anti-neoplastic agents can lead to the development of chemotherapy induced peripheral neuropathy (CIPN), which is long lasting and often refractory to treatment. This neuropathic pain develops along dermatomes innervated by peripheral nerves with cell bodies located in the dorsal root ganglia (DRG). The voltage-gated sodium channel NaV1.7 is expressed at high levels in peripheral nerve tissues and has been implicated in the development of CIPN. Efforts to develop novel analgesics directly inhibiting NaV1.7 have been unsuccessful, and our group has pioneered an alternative approach based on indirect modulation of channel trafficking by the accessory protein collapsin response mediator protein 2 (CRMP2). We have recently reported a small molecule, compound 194, that inhibits CRMP2 SUMOylation by the E2 SUMO-conjugating enzyme Ubc9 (Cai et al. , Sci. Transl. Med. 2021 13(619):eabh1314). Compound 194 is a potent and selective inhibitor of NaV1.7 currents in DRG neurons and reverses mechanical allodynia in models of surgical, inflammatory, and neuropathic pain, including spared nerve injury and paclitaxelinduced peripheral neuropathy. Here we report that, in addition to its reported effects in rats, 194 also reduces mechanical allodynia in male CD-1 mice treated with platinumcomplex agent oxaliplatin. Importantly, treatment with 194 prevented the development of mechanical allodynia when co-administered with oxaliplatin. No effects were observed on the body weight of animals treated with oxaliplatin or 194 throughout the study period. These findings support the notion that 194 is a robust inhibitor of CIPN that reduces established neuropathic pain and prevents the emergence of neuropathic pain during treatment with multiple anti-neoplastic agents in both mice and rats.

Pharmacologic Inhibition of SUMO-Activating Enzyme Potentiates Interferon Response and T Cell-Mediated Anti-Tumor Immunity in Chronic Lymphocytic Leukemia (CLL) and Lymphoma Models

Introduction: CLL is characterized by deficient immunity which clinically manifests as increased predisposition towards malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFκB signaling, IFN response and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL.Methods: T cells from patients with CLL were purified using Dynabeads. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP; Clariom S), RNA was harvested after 3 and 24 hours of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 µg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days prior to spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1x10 6 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA).Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement (αCD3/CD28). Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFκB and NFAT activation detected by immunoblotting, immunocytochemistry and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4 + T cells following 72 and 96 hours of TCR stimulation vs. control. Meanwhile, expression of CD25, HLA-DR and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced two-fold in TAK-981-treated cells after 24 hours and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 μM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL-2. However, T cells did not undergo apoptosis under those conditions.Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNγ and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift towards Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4 + naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNγ and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets.Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in expression of FoxP3 and an increased production of IFNγ (Figure 1). In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNγ secretion in CD8+ TILs.Conclusion. Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. [Display omitted] DisclosuresSiddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Danilov: SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding.

Staphylococcus aureus Decreases SUMOylation Host Response to Promote Intramacrophage Survival.

Staphylococcus aureus is a commensal bacterium that causes severe infections in soft tissue and the bloodstream. During infection, S. aureus manipulates host cell response to facilitate its own replication and dissemination. Here, we show that S. aureus significantly decreases the level of SUMOylation, an essential post-translational modification, in infected macrophages 24 h post-phagocytosis. The reduced level of SUMOylation correlates with a decrease in the SUMO-conjugating enzyme Ubc9. The over-expression of SUMO proteins in macrophages impaired bacterial intracellular proliferation and the inhibition of SUMOylation with ML-792 increased it. Together, these findings demonstrated for the first time the role of host SUMOylation response toward S. aureus infection.

Poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis

ABSTRACT SUMO modification is required for the kinetochore localization of the kinesin-like motor protein CENP-E, which subsequently mediates the alignment of chromosomes to the spindle equator during mitosis. However, the underlying mechanisms by which sumoylation regulates CENP-E kinetochore localization are still unclear. In this study, we first elucidate that the kinetochore protein Nuf2 is not only required for CENP-E kinetochore localization but also preferentially modified by poly-SUMO-2/3 chains. In addition, poly-SUMO-2/3 modification of Nuf2 is significantly upregulated during mitosis, which is temporally correlated to the kinetochore localization of CENP-E during mitosis. We further show that the mitotic defects in CENP-E kinetochore localization and chromosome congression caused by global inhibition of sumoylation can be rescued by expressing a fusion protein between Nuf2 and the SUMO-conjugating enzyme Ubc9 for stimulating Nuf2 SUMO-2/3 modification. Moreover, the expression of another fusion protein between Nuf2 and three SUMO-2 moieties (SUMO-2 trimer), which mimics the trimeric SUMO-2/3 chain modification of Nuf2, can also rescue the mitotic defects due to global inhibition of sumoylation. Conversely, expressing the other forms of Nuf2-SUMO fusion proteins, which imitate Nuf2 modifications by SUMO-2/3 monomer, SUMO-2/3 dimer, and SUMO-1 trimer, respectively, cannot rescue the same mitotic defects. Lastly, compared to Nuf2, the fusion protein simulating the trimeric SUMO-2 chain-modified Nuf2 exhibits a significantly higher binding affinity to CENP-E wild type containing a functional SUMO-interacting motif (SIM) but not the CENP-E SIM mutant. Hence, our results support a model that poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis. Abbreviations: CENP-E, centromere-associated protein E; SUMO, small ubiquitin-related modifier; SIM, SUMO-interacting motif.

Sumoylation of the Carboxy-Terminal of Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Attenuates Viral DNA Replication.

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.

SUMOylation of the transcription factor ZFHX3 at Lys-2806 requires SAE1, UBC9, and PIAS2 and enhances its stability and function in cell proliferation

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

UBC-9 Acts in GABA Neurons to Control Neuromuscular Signaling in C. elegans.

Regulation of excitatory to inhibitory signaling balance is essential to nervous system health and is maintained by numerous enzyme systems that modulate the activity, localization, and abundance of synaptic proteins. SUMOylation is a key post-translational regulator of protein function in diverse cells, including neurons. There, its role in regulating synaptic transmission through pre- and postsynaptic effects has been shown primarily at glutamatergic central nervous system synapses, where the sole SUMO-conjugating enzyme Ubc9 is a critical player. However, whether Ubc9 functions globally at other synapses, including inhibitory synapses, has not been explored. Here, we investigated the role of UBC-9 and the SUMOylation pathway in controlling the balance of excitatory cholinergic and inhibitory GABAergic signaling required for muscle contraction in Caenorhabditis elegans. We found inhibition or overexpression of UBC-9 in neurons modestly increased muscle excitation. Similar and even stronger phenotypes were seen with UBC-9 overexpression specifically in GABAergic neurons, but not in cholinergic neurons. These effects correlated with accumulation of synaptic vesicle-associated proteins at GABAergic presynapses, where UBC-9 and the C. elegans SUMO ortholog SMO-1 localized, and with defects in GABA-dependent behaviors. Experiments involving expression of catalytically inactive UBC-9 [UBC-9(C93S)], as well as co-expression of UBC-9 and SMO-1, suggested wild type UBC-9 overexpressed alone may act via substrate sequestration in the absence of sufficient free SUMO, underscoring the importance of tightly regulated SUMO enzyme function. Similar effects on muscle excitation, GABAergic signaling, and synaptic vesicle localization occurred with overexpression of the SUMO activating enzyme subunit AOS-1. Together, these data support a model in which UBC-9 and the SUMOylation system act at presynaptic sites in inhibitory motor neurons to control synaptic signaling balance in C. elegans. Future studies will be important to define UBC-9 targets at this synapse, as well as mechanisms by which UBC-9 and the SUMO pathway are regulated.

SUMOylation is required for PIPK1γ-driven keratinocyte migration and growth.

PIPKIγ, a key member of the type I phosphatidylinositol 4-phosphate kinase (PIPKI) family that regulates the spatial-temporal generation of PIP2, has been implicated in diverse biological processes including cell survival, cell polarity, and cell migration. An essential role of PIPKIγ in tumor cells and nerve cells has been established in previous studies. However, the function and regulatory mechanism of PIPKIγ remains incompletely understood. Here, we showed that PIPKIγ can specifically associate with the SUMO-conjugating (E2) enzyme UBC9 and can be SUMOylated both invivo and invitro. We further identified that Lys-591 is the critical SUMO-acceptor site of PIPKIγ and that SUMO conjugation at this site is required for PIPKIγ-driven keratinocyte migration and growth. Mechanistically, SUMOylation deficiency significantly disrupts PIPKIγ-mediated generation of intracellular PIP2, rather than the subcellular translocation and protein stability of PIPKIγ. Our findings reveal that PIPKIγ is a novel SUMOylation target and highlight the essential role of PIPKIγ SUMOylation in human keratinocyte function, providing an important orientation for in-depth study of wound repair.

The synaptic balance between sumoylation and desumoylation is maintained by the activation of metabotropic mGlu5 receptors.

Sumoylation is a reversible post-translational modification essential to the modulation of neuronal function, including neurotransmitter release and synaptic plasticity. A tightly regulated equilibrium between the sumoylation and desumoylation processes is critical to the brain function and its disruption has been associated with several neurological disorders. This sumoylation/desumoylation balance is governed by the activity of the sole SUMO-conjugating enzyme Ubc9 and a group of desumoylases called SENPs, respectively. We previously demonstrated that the activation of type 5 metabotropic glutamate receptors (mGlu5R) triggers the transient trapping of Ubc9 in dendritic spines, leading to a rapid increase in the overall synaptic sumoylation. However, the mechanisms balancing this increased synaptic sumoylation are still not known. Here, we examined the diffusion properties of the SENP1 enzyme using a combination of advanced biochemical approaches and restricted photobleaching/photoconversion of individual hippocampal spines. We demonstrated that the activation of mGlu5R leads to a time-dependent decrease in the exit rate of SENP1 from dendritic spines. The resulting post-synaptic accumulation of SENP1 restores synaptic sumoylation to initial levels. Altogether, our findings reveal the mGlu5R system as a central activity-dependent mechanism to maintaining the homeostasis of sumoylation at the mammalian synapse.

Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

Epstein-Barr Virus latent membrane protein-1 (LMP1) interacts with the SUMO-conjugating enzyme Ubc9, which induces protein sumoylation and may contribute to LMP1-mediated oncogenesis. After analyzing human lymphoma tissues and EBV-positive cell lines, we now document a strong correlation between LMP1 and sumo-1/2/3 or SUMO-1/2/3 levels, and show that LMP1-induced sumo expression requires the activation of NF-κB signaling through CTAR1 and CTAR2. Together, these results point to a second mechanism by which LMP1 dysregulates sumoylation processes and adds EBV-associated lymphomas to the list of malignancies associated with increased SUMO expression.