Auto-sumoylation of the Ubc9 E2 SUMO-conjugating Enzyme Extends Cellular Lifespan.

Calorie restriction (CR) provides anti-aging benefits through diverse processes, such as reduced metabolism and growth and increased mitochondrial activity. Although controversy still exists regarding CR-mediated lifespan effects, many researchers are seeking interventions that mimic the effects of CR. Yeast has proven to be a useful model system for aging studies, including CR effects. We report here that yeast adapted through in vitro evolution to the severe cellular stress caused by loss of the Ulp2 SUMO-specific protease exhibit both enhanced growth rates and replicative lifespan, and they have altered gene expression profiles similar to those observed in CR. Notably, in certain evolved ulp2Δ lines, a dramatic increase in the auto-sumoylation of Ubc9 E2 SUMO-conjugating enzyme results in altered regulation of multiple targets involved in energy metabolism and translation at both transcriptional and post-translational levels. This increase is essential for the survival of aged cells and CR-mediated lifespan extension. Thus, we suggest that high Ubc9 auto-sumoylation exerts potent anti-aging effects by promoting efficient energy metabolism-driven improvements in cell replication abilities. This potential could be therapeutically explored for the development of novel CR-mimetic strategies.

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

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.

UBC9-mediated SUMOylation of Lamin B1 enhances DNA-damage-induced nuclear DNA leakage and autophagy after spinal cord injury.

Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI). Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence. In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death. Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.

Mechanisms and functions of SUMOylation in health and disease: a review focusing on immune cells.

SUMOylation, which is a type of post-translational modification that involves covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to target substrates, regulates various important molecular and cellular processes, including transcription, the cell cycle, cell signaling, and DNA synthesis and repair. Newly synthesized SUMO is immature and cleaved by the SUMO-specific protease family, resulting in exposure of the C-terminal Gly-Gly motif to become the mature form. In the presence of ATP, mature SUMO is conjugated with the activating enzyme E1 through the cysteine residue of E1, followed by transfer to the cysteine residue of E2-conjugating enzyme Ubc9 in humans that recognizes and modifies the lysine residue of a substrate protein. E3 SUMO ligases promote SUMOylation. SUMOylation is a reversible modification and mediated by SUMO-specific proteases. Cumulative studies have indicated that SUMOylation affects the functions of protein substrates in various manners, including cellular localization and protein stability. Gene knockout studies in mice have revealed that several SUMO cycling machinery proteins are crucial for the development and differentiation of various cell lineages, including immune cells. Aberrant SUMOylation has been implicated in several types of diseases, including cancers, cardiovascular diseases, and autoimmune diseases. This review summarizes the biochemistry of SUMO modification and the general biological functions of proteins involved in SUMOylation. In particular, this review focuses on the molecular mechanisms by which SUMOylation regulates the development, maturation, and functions of immune cells, including T, B, dendritic, and myeloid cells. This review also discusses the underlying relevance of disruption of SUMO cycling and site-specific interruption of SUMOylation on target proteins in immune cells in diseases, including cancers and infectious diseases.

Ubiquitin-conjugating enzyme E2 for regulating autophagy in diabetic cardiomyopathy: A mini-review.

The prevalence of diabetic cardiomyopathy (DCM) increases year by year with the increase in the prevalence of diabetes mellitus (DM), which is one of the most serious cardiovascular complications of DM and a major cause of death in diabetic patients. Although the pathological molecular features of DCM have not been fully elucidated, increasing evidence suggests that impaired autophagy in cardiomyocytes plays a nonnegligible role in the development of DCM. It has been shown that SUMOylation [SUMO = small ubiquitin-like modifier], a post-translational modification of proteins, and its associated ubiquitin-proteasome system mediates protein quality control in the heart and plays an important role in the proteotoxic environment of the heart. Specifically, the expression of ubiquitin-conjugating enzyme E2 (Ubc9), the only SUMO-E2 enzyme, exerts a positive regulatory effect on autophagy in cardiomyocytes with potential cardioprotective effects. This review focuses on the role that autophagy plays in DCM and the potential for Ubc9-regulated autophagy pathways to ameliorate DCM, highlighting the potential of Ubc9 as an interventional target in DCM and providing new insights into the pathogenesis of the disease.

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.

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-ß 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-ß 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-ß 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-ß 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.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

A novel cell-permeable peptide prevents protein SUMOylation and supports the mislocalization and aggregation of TDP-43.

SUMOylation is a post-translational modification (PTM) that exerts a regulatory role in different cellular processes, including protein localization, aggregation, and biological activities. It consists of the dynamic formation of covalent isopeptide bonds between a family member of the Small Ubiquitin Like Modifiers (SUMOs) and the target proteins. Interestingly, it is a cellular mechanism implicated in several neurodegenerative pathologies and potentially it could become a new therapeutic target; however, there are very few pharmacological tools to modulate the SUMOylation process. In this study, we have designed and tested the activity of a novel small cell-permeable peptide, COV-1, in a neuroblastoma cell line that specifically prevents protein SUMOylation. COV-1 inhibits UBC9-protein target interaction and efficiently decreases global SUMO-1ylation. Moreover, it can perturb RanGAP-1 perinuclear localization by inducing the downregulation of UBC9. In parallel, we found that COV-1 causes an increase in the ubiquitin degradation system up to its engulfment while enhancing the autophagic flux. Surprisingly, COV-1 modifies protein aggregation, and specifically it mislocalizes TDP-43 within cells, inducing its aggregation and co-localization with SUMO-1. These data suggest that COV-1 could be taken into future consideration as an interesting pharmacological tool to study the cellular cascade effects of SUMOylation prevention.

Global SUMOylation in mouse oocytes maintains oocyte identity and regulates chromatin remodeling and transcriptional silencing at the end of folliculogenesis.

Meiotically competent oocytes in mammals undergo cyclic development during folliculogenesis. Oocytes within ovarian follicles are transcriptionally active, producing and storing transcripts required for oocyte growth, somatic cell communication and early embryogenesis. Transcription ceases as oocytes transition from growth to maturation and does not resume until zygotic genome activation. Although SUMOylation, a post-translational modification, plays multifaceted roles in transcriptional regulation, its involvement during oocyte development remains poorly understood. In this study, we generated an oocyte-specific knockout of Ube2i, encoding the SUMO E2 enzyme UBE2I, using Zp3-cre+ to determine how loss of oocyte SUMOylation during folliculogenesis affects oocyte development. Ube2i Zp3-cre+ female knockout mice were sterile, with oocyte defects in meiotic competence, spindle architecture and chromosome alignment, and a premature arrest in metaphase I. Additionally, fully grown Ube2i Zp3-cre+ oocytes exhibited sustained transcriptional activity but downregulated maternal effect genes and prematurely activated genes and retrotransposons typically associated with zygotic genome activation. These findings demonstrate that UBE2I is required for the acquisition of key hallmarks of oocyte development during folliculogenesis, and highlight UBE2I as a previously unreported orchestrator of transcriptional regulation in mouse oocytes.

Structural insights into the regulation of the human E2∼SUMO conjugate through analysis of its stable mimetic.

Protein SUMOylation is a ubiquitylation-like post-translational modification (PTM) that is synthesized through an enzymatic cascade involving an E1 (SAE1:SAE2), an E2 (UBC9), and various E3 enzymes. In the final step of this process, the small ubiquitin-like modifier (SUMO) is transferred from the UBC9∼SUMO thioester onto a lysine residue of a protein substrate. This reaction can be accelerated by an E3 ligase. As the UBC9∼SUMO thioester is chemically unstable, a stable mimetic is desirable for structural studies of UBC9∼SUMO alone and in complex with a substrate and/or an E3 ligase. Recently, a strategy for generating a mimetic of the yeast E2∼SUMO thioester by mutating alanine 129 of Ubc9 to a lysine has been reported. Here, we reproduce and further investigate this approach using the human SUMOylation system and characterize the resulting mimetic of human UBC9∼SUMO1. We show that substituting lysine for alanine 129, but not for other active-site UBC9 residues, results in a UBC9 variant that is efficiently auto-SUMOylated. The auto-modification is dependent on cysteine 93 of UBC9, suggesting that it proceeds via this residue, through the same pathway as that for SUMOylation of substrates. The process is also partially dependent on aspartate 127 of UBC9 and accelerated by high pH, highlighting the importance of the substrate lysine protonation state for efficient SUMOylation. Finally, we present the crystal structure of the UBC9-SUMO1 molecule, which reveals the mimetic in an open conformation and its polymerization via the noncovalent SUMO-binding site on UBC9. Similar interactions could regulate UBC9∼SUMO in some cellular contexts.

The Rosetta Stone Hypothesis-Based Interaction of the Tumor Suppressor Proteins Nit1 and Fhit.

In previous studies, we have identified the tumor suppressor proteins Fhit (fragile histidine triad) and Nit1 (Nitrilase1) as interaction partners of ß-catenin both acting as repressors of the canonical Wnt pathway. Interestingly, in D. melanogaster and C. elegans these proteins are expressed as NitFhit fusion proteins. According to the Rosetta Stone hypothesis, if proteins are expressed as fusion proteins in one organism and as single proteins in others, the latter should interact physically and show common signaling function. Here, we tested this hypothesis and provide the first biochemical evidence for a direct association between Nit1 and Fhit. In addition, size exclusion chromatography of purified recombinant human Nit1 showed a tetrameric structure as also previously observed for the NitFhit Rosetta Stone fusion protein Nft-1 in C. elegans. Finally, in line with the Rosetta Stone hypothesis we identified Hsp60 and Ubc9 as other common interaction partners of Nit1 and Fhit. The interaction of Nit1 and Fhit may affect their enzymatic activities as well as interaction with other binding partners.

Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast.

Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.

SUMOylation of ERp44 enhances Ero1α ER retention contributing to the pathogenesis of obesity and insulin resistance.

OBJECTIVE: As the only E2 conjugating enzyme for the SUMO system, Ubc9-mediated SUMOylation has been recognized to regulate diverse biological processes, but its impact on adipocytes relevant to obesity and insulin resistance is yet to be elucidated. METHODS: We established adipocyte-specific Ubc9 deficient mice to explore the effects of Ubc9 on obesity and metabolic disorders induced by high-fat diet (HFD) in adult mice. The molecular targets of SUMOylation were explored by liquid chromatography-mass spectrometry, and the regulatory mechanism of SUMOylation in T2D was analyzed. RESULTS: Adipocyte-specific depletion of Ubc9 (AdipoQ-Cre-Ubc9fl/fl, Ubc9AKO) protected mice from HFD-induced obesity, insulin resistance, and hepatosteatosis. The Ubc9AKO mice were featured by the reduced HFD-induced endoplasmic reticulum (ER) stress and inflammatory response. Mechanically, over nutrition rendered adipocytes to undergo a SUMOylation turnover characterized by the change of SUMOylation levels and substrates. ERp44 displayed the highest change in terms of SUMOylation levels of substrates involved in ER-related functions. The lack of ERp44 SUMOylation at lysine 76 (K76) located within the thioredoxin (TRX)-like domain by Ubc9 deficiency enhanced its degradation and suppressed its covalent binding to Ero1α, an oxidase that exists in the ER but lacks ER retention motif, thereby alleviating endoplasmic reticulum stress by promoting Ero1α secretion. CONCLUSIONS: Our data suggest that modulation of ERp44 SUMOylation in adipocytes could be a feasible strategy against obesity and insulin resistance in clinical settings.

In vitro and in cellulae methods for determining the target protein SUMOylation.

Post-translational modifications (PTMs) provide a critical means of calibrating the functional proteome and, thus, are extensively utilized by the eukaryotes to exert spatio-temporal regulation on the cellular machinery rapidly. Ubiquitination and phosphorylation are examples of the well-documented PTMs. SUMOylation, the reversible conjugation of the Small Ubiquitin-related MOdifier (SUMO) at a specific lysine residue on a target protein, bears striking similarity with ubiquitination and follows an enzymatic cascade for the attachment of SUMO to the target protein. Unlike Ubiquitination, SUMOylation can modulate the target protein's structure, stability, activity, localization, and interaction. Thus, SUMOylation regulates cellular events such as signal transduction, cell-cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Accordingly, deregulation of SUMOylation is an avenue for diseases, which makes the investigation of SUMO and its substrates within the cell essential. However, the low extent of SUMOylation has posed a significant challenge in detecting SUMO modification within the cell. Bioinformatics tools can help predict SUMOylation, and mass-spectrometric analysis can identify a pool of cellular protein SUMOylome. Nevertheless, the biochemical methods for observing the enhanced level of in vitro SUMOylation help validate protein SUMOylation, critical lysine(s) utilized in the process, and its effect on substrate protein function. This chapter provides a detailed account of biochemical methods commonly utilized to detect SUMOylated proteins that are central for understanding the biological functions and mechanism of regulation of SUMO targets.

The UBC9/SUMO pathway affects E-cadherin cleavage in HPV-positive head and neck cancer.

Functional loss of E-cadherin is frequent during tumor progression and occurs through a variety of mechanisms, including proteolytic cleavage. E-cadherin downregulation leads to the conversion of a more malignant phenotype promoting Epithelial to Mesenchymal Transition (EMT). The UBC9/SUMO pathway has been also shown to be involved in the regulation of EMT in different cancers. Here we found an increased expression of UBC9 in the progression of Head and Neck Cancer (HNC) and uncovered a role for UBC9/SUMO in hampering the HPV-mediated E-cadherin cleavage in HNC.

Abeta-induced presynaptic release of UBC9 through extracellular vesicles involves SNAP23.

Ubiquitin conjugating enzyme 9 (UBC9), the sole small ubiquitin-like modifier(SUMO) conjugating enzyme, is considered to be a vital regulator of the mechanism of SUMOylation and is likely to participate in the progression of Alzheimer's disease (AD). Our previous studies found that UBC9 is highly mobile in neurons, but the underlying mechanism is still unknown. We designed to investigate the underlying mechanism of the synaptic redistribution of UBC9 in AD. We found that ß-amyloid peptide (Aß) significantly decreased presynaptic UBC9 expression and increased postsynaptic UBC9 expression but did not affect UBC9 expression in synaptosomes. Moreover, there is evidence that extracellular vesicles (EVs) may facilitate synaptic gap transmission. Immunoprecipitation assays showed that flotillin, which is specifically expressed in EVs, co-immunoprecipitated with UBC9 in cell lysates and media and that Aß treatment enhanced this interaction. Additionally, epidermal growth factor and oleanolic acid have been found to either promote or inhibit the release of EVs, thus blocking the Aß-induced presynaptic release of UBC9. However, knockdown of synaptosome associated protein 23 (SNAP-23) or inhibition of SNAP23 phosphorylation was found to secure Aß-induced presynaptic release of UBC9. These results suggest that Aß induces redistribution of UBC9 from presynaptic to postsynaptic terminals, which may mediate through EVs associated with SNAP23. Our study reveals the cellular mechanisms of EVs as an essential component of the presynaptic release of UBC9.

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.

A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells.

BACKGROUND: Mantle cell lymphoma (MCL) is a rare, highly heterogeneous type of B-cell non-Hodgkin's lymphoma. The sumoylation pathway is known to be upregulated in many cancers including lymphoid malignancies. However, little is known about its oncogenic role in MCL. METHODS: Levels of sumoylation enzymes and sumoylated proteins were quantified in MCL cell lines and primary MCL patient samples by scRNA sequencing and immunoblotting. The sumoylation enzyme SAE2 was genetically and pharmacologically targeted with shRNA and TAK-981 (subasumstat). The effects of SAE2 inhibition on MCL proliferation and cell cycle were evaluated using confocal microscopy, live-cell microscopy, and flow cytometry. Immunoprecipitation and orbitrap mass spectrometry were used to identify proteins targeted by sumoylation in MCL cells. RESULTS: MCL cells have significant upregulation of the sumoylation pathway at the level of the enzymes SAE1 and SAE2 which correlated with poor prognosis and induction of mitosis associated genes. Selective inhibition of SAE2 with TAK-981 results in significant MCL cell death in vitro and in vivo with mitotic dysregulation being an important mechanism of action. We uncovered a sumoylation program in mitotic MCL cells comprised of multiple pathways which could be directly targeted with TAK-981. Centromeric localization of topoisomerase 2A, a gene highly upregulated in SAE1 and SAE2 overexpressing MCL cells, was lost with TAK-981 treatment likely contributing to the mitotic dysregulation seen in MCL cells. CONCLUSIONS: This study not only validates SAE2 as a therapeutic target in MCL but also opens the door to further mechanistic work to uncover how to best use desumoylation therapy to treat MCL and other lymphoid malignancies.

The B-box1 domain of PML mediates SUMO E2-E3 complex formation through an atypical interaction with UBC9.

The small, ubiquitin-like modifier SUMO is covalently attached to substrates by the enzyme UBC9. SUMO conjugation of substrates often requires an E3 ligase, which ensures substrate specificity by simultaneously binding UBC9 and the substrate. E3 SUMO ligases commonly use a RING domain to engage UBC9. The Promyelocytic Leukemia protein (PML) has been implicated as a probable SUMO ligase. Although PML does contain a RING domain, which is expected to recruit UBC9, we demonstrate that PML RING does not bind UBC9 in vitro. Instead, we show that isolated PML B-box1 possesses UBC9-binding activity and map the B-box1 binding site on UBC9. This site also binds the upstream E1 enzyme that transfers SUMO to UBC9. The overlap of these two binding sites suggests that UBC9 cannot interact with its E1 and E3 partners simultaneously. Furthermore, we present a model of the PML dimer that supports the accessibility of B-box1 for UBC9 binding in the context of the full-length PML.