SUMO Pathway Research Articles

The Role of Ubiquitination and SUMOylation in Telomere Biology.

Telomeres are a unique structure of DNA repeats covered by proteins at the ends of the chromosomes that protect the coding regions of the genome and function as a biological clock. They require a tight regulation of the factors covering and protecting their structure, as they are shortened with each cell division to limit the ability of cells to replicate uncontrollably. Additionally, they protect the chromosome ends from DNA damage responses and thereby, prevent genomic instability. Telomere dysfunction can lead to chromosomal abnormalities and cancer. Therefore, dysregulation of any of the factors that regulate the integrity of the telomeres will have implications to chromosomal stability, replicative lifespan and may lead to cell transformation. This review will cover the main factors participating in the normal function of the telomeres and how these are regulated by the ubiquitin and SUMO systems. Accumulating evidence indicate that the ubiquitin and SUMO pathways are significant regulators of the shelterin complex and other chromatin modifiers, which are important for telomere structure integrity. Furthermore, the crosstalk between these two pathways has been reported in telomeric DNA repair. A better understanding of the factors contributing to telomere biology, and how they are regulated, is important for the design of new strategies for cancer therapies and regenerative medicine.

Ubiquitination and SUMOylation in HIV Infection: Friends and Foes.

As intracellular parasites, viruses hijack the cellular machinery to facilitate their replication and spread. This includes favouring the expression of their viral genes over host genes, appropriation of cellular molecules, and manipulation of signalling pathways, including the post-translational machinery. HIV, the causative agent of AIDS, is notorious for using post-translational modifications to generate infectious particles. Here, we discuss the mechanisms by which HIV usurps the ubiquitin and SUMO pathways to modify both viral and host factors to achieve a productive infection, and also how the host innate sensing system uses these post-translational modifications to hinder HIV replication.

Developmental profiles of SUMOylation pathway proteins in rat cerebrum and cerebellum.

Protein SUMOylation regulates multiple processes involved in the differentiation and maturation of cells and tissues during development. Despite this, relatively little is known about the spatial and temporal regulation of proteins that mediate SUMOylation and deSUMOylation in the CNS. Here we monitor the expression of key SUMO pathway proteins and levels of substrate protein SUMOylation in the forebrain and cerebellum of Wistar rats during development. Overall, the SUMOylation machinery is more highly-expressed at E18 and decreases thereafter, as previously described. All of the proteins investigated are less abundant in adult than in embryonic brain. Furthermore, we show for first time that the profiles differ between cerebellum and cerebrum, indicating differential regional regulation of some of the proteins analysed. These data provide further basic observation that may open a new perspective of research about the role of SUMOylation in the development of different brain regions.

The SUMO-Conjugase Ubc9 Prevents the Degradation of the Dopamine Transporter, Enhancing Its Cell Surface Level and Dopamine Uptake.

The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson’s disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.

SUMOylation by SUMO2 is implicated in the degradation of misfolded ataxin-7 via RNF4 in SCA7 models.

ABSTRACTPerturbation of protein homeostasis and aggregation of misfolded proteins is a major cause of many human diseases. A hallmark of the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7) is the intranuclear accumulation of mutant, misfolded ataxin-7 (polyQ-ATXN7). Here, we show that endogenous ATXN7 is modified by SUMO proteins, thus also suggesting a physiological role for this modification under conditions of proteotoxic stress caused by the accumulation of polyQ-ATXN7. Co-immunoprecipitation experiments, immunofluorescence microscopy and proximity ligation assays confirmed the colocalization and interaction of polyQ-ATXN7 with SUMO2 in cells. Moreover, upon inhibition of the proteasome, both endogenous SUMO2/3 and the RNF4 ubiquitin ligase surround large polyQ-ATXN7 intranuclear inclusions. Overexpression of RNF4 and/or SUMO2 significantly decreased levels of polyQ-ATXN7 and, upon proteasomal inhibition, led to a marked increase in the polyubiquitination of polyQ-ATXN7. This provides a mechanism for the clearance of polyQ-ATXN7 from affected cells that involves the recruitment of RNF4 by SUMO2/3-modified polyQ-ATXN7, thus leading to its ubiquitination and proteasomal degradation. In a SCA7 knock-in mouse model, we similarly observed colocalization of SUMO2/3 with polyQ-ATXN7 inclusions in the cerebellum and retina. Furthermore, we detected accumulation of SUMO2/3 high-molecular-mass species in the cerebellum of SCA7 knock-in mice, compared with their wild-type littermates, and changes in SUMO-related transcripts. Immunohistochemical analysis showed the accumulation of SUMO proteins and RNF4 in the cerebellum of SCA7 patients. Taken together, our results show that the SUMO pathway contributes to the clearance of aggregated ATXN7 and suggest that its deregulation might be associated with SCA7 disease progression.

Fish SUMO3 functions as a critical antiviral molecule against iridovirus and nodavirus

Protein SUMOylation (SUMO is small ubiquitin-related modifier) is a dynamic process that is strictly regulated under physiological and pathological conditions. We previously cloned and characterized two SUMO homologue genes (EcSUMO1 and EcSUMO2) from orange-spotted grouper (Epinephelus coioides). In the present study, the SUMO3 homologue from E. coioides (EcSUMO3) was cloned and its possible roles in fish immunity were analyzed. The open reading frame of EcSUMO3 contains 285 base pairs encoding a 94 amino acid protein with a predicted molecular mass of 10.73 kDa. The protein sequence of EcSUMO3 revealed similar domains with mammals, including the UBQ (ubiquitin-like proteins) domain, the hydrophobic surface, the Ulp1-Smt3 interaction sites, a VKTE motif and the C-terminal Gly residues. EcSUMO3 shares 46.83% and 89.58% identity with EcSUMO1 and EcSUMO2, respectively, and it shares 94%, 98%, and 98% identity with SUMO3 from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. Quantitative real-time polymerase chain reaction analysis indicated that EcSUMO3 was constitutively expressed in all of the analyzed tissues in healthy grouper. EcSUMO3 expression levels were remarkably (p < 0.01) up-regulated in grouper spleen (GS) cells in response to stimulation with red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV). EcSUMO3 was distributed in both the cytoplasm and nucleus in GS cells. EcSUMO3 enhanced SGIV and RGNNV replication during viral infection in vitro. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.

Binding to small ubiquitin-like modifier and the nucleolar protein Csm1 regulates substrate specificity of the Ulp2 protease

Ulp1 and Ulp2, in the yeast Saccharomyces cerevisiae, are the founding members of deSUMOylating enzymes. These enzymes remove small ubiquitin-like modifier (SUMO) from proteins and are conserved in all eukaryotes. Previous studies have shown that Ulp1 deSUMOylates the bulk of intracellular SUMOylated proteins, whereas Ulp2 is a highly specific enzyme. However, the mechanism for Ulp2's substrate specificity has been insufficiently understood. Here we show that the C-terminal regulatory domain of Ulp2 contains three distinct, yet conserved, motifs that control its in vivo substrate specificity and cell growth. Among them, a SUMO-interacting motif (SIM) was found to coordinate with the domain of Ulp2 that binds to the nucleolar protein Csm1 to ensure maximal deSUMOylation of Ulp2's nucleolar substrates. We found that whereas the Csm1-binding domain of Ulp2 recruits this enzyme to the nucleolus, Ulp2's C-terminal SIM promotes its SUMO protease activity and plays a key role in mediating the in vivo specificity of Ulp2. Thus, the substrate specificity of Ulp2 is controlled by both its subcellular localization and the SUMOylation status of its substrates. These findings illustrate the highly coordinated and dynamic nature of the SUMO pathways in maintaining homeostasis of intracellular SUMOylation.

Targeting the SUMO Pathway Primes All-trans Retinoic Acid–Induced Differentiation of Nonpromyelocytic Acute Myeloid Leukemias

Differentiation therapies using all-trans retinoic acid (ATRA) are highly efficient at treating acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, their efficacy, if any, is limited in the case of non-APL AML. We report here that inhibition of SUMOylation, a posttranslational modification related to ubiquitination, restores the prodifferentiation and antiproliferative activities of retinoids in non-APL AML. Controlled inhibition of SUMOylation with the pharmacologic inhibitors 2-D08 or anacardic acid, or via overexpression of SENP deSUMOylases, enhanced the ATRA-induced expression of key genes involved in differentiation, proliferation, and apoptosis in non-APL AML cells. This activated ATRA-induced terminal myeloid differentiation and reduced cell proliferation and viability, including in AML cells resistant to chemotherapeutic drugs. Conversely, enhancement of SUMOylation via overexpression of the SUMO-conjugating enzyme Ubc9 dampened expression of ATRA-responsive genes and prevented differentiation. Thus, inhibition of the SUMO pathway is a promising strategy to sensitize patients with non-APL AML to retinoids and improve the treatment of this poor-prognosis cancer.Significance: SUMOylation silences key ATRA-responsive genes in nonpromyelocytic acute myeloid leukemias. Cancer Res; 78(10); 2601-13. ©2018 AACR.

Visualization of human karyopherin beta-1/importin beta-1 interactions with protein partners in mitotic cells by co-immunoprecipitation and proximity ligation assays

Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells. By combining co-immunoprecipitation and proteome-wide mass spectrometry analysis of synchronized cell extracts, we identified expected (e.g., RAN and SUMO pathway factors) and novel mitotic interactors of importin beta-1, many with RNA-binding ability, that had not been previously associated with importin beta-1. These data complement interactomic studies of interphase transport pathways. We further developed automated proximity ligation assay (PLA) protocols to validate selected interactors. We succeeded in obtaining spatial and temporal resolution of genuine importin beta-1 interactions, which were visualized and localized in situ in intact mitotic cells. Further developments of PLA protocols will be helpful to dissect importin beta-1-orchestrated pathways during mitosis.

Failed mitochondrial import and impaired proteostasis trigger SUMOylation of mitochondrial proteins

Modification by the ubiquitin-like protein SUMO affects hundreds of cellular substrate proteins and regulates a wide variety of physiological processes. While the SUMO system appears to predominantly target nuclear proteins and, to a lesser extent, cytosolic proteins, hardly anything is known about the SUMOylation of proteins targeted to membrane-enclosed organelles. Here, we identify a large set of structurally and functionally unrelated mitochondrial proteins as substrates of the SUMO pathway in yeast. We show that SUMO modification of mitochondrial proteins does not rely on mitochondrial targeting and, in fact, is strongly enhanced upon import failure, consistent with the modification occurring in the cytosol. Moreover, SUMOylated forms of mitochondrial proteins particularly accumulate in HSP70- and proteasome-deficient cells, suggesting that SUMOylation participates in cellular protein quality control. We therefore propose that SUMO serves as a mark for nonfunctional mitochondrial proteins, which only sporadically arise in unstressed cells but strongly accumulate upon defective mitochondrial import and impaired proteostasis. Overall, our findings provide support for a role of SUMO in the cytosolic response to aberrant proteins.

Targeting the SUMO pathway as a novel treatment for anaplastic thyroid cancer.

Cancer stem cells (CSCs) are expanded in anaplastic thyroid cancer (ATC) and standard treatment approaches have failed to improve survival, suggesting a need to specifically target the CSC population. Recent studies in breast and colorectal cancer demonstrated that inhibition of the SUMO pathway repressed CD44 and cleared the CSC population, mediated through SUMO-unconjugated TFAP2A. We sought to evaluate effects of inhibiting the SUMO pathway in ATC. ATC cell lines and primary ATC tumor samples were evaluated. The SUMO pathway was inhibited by knockdown of PIAS1 and use of SUMO inhibitors anacardic acid and PYR-41. The expression of TFAP2A in primary ATC was examined by immunohistochemistry. All ATC cell lines expressed TFAP2A but only 8505C expressed SUMO-conjugated TFAP2A. In 8505C only, inhibition of the SUMO pathway by knockdown of PIAS1 or treatment with SUMO inhibitors repressed expression of CD44 with a concomitant loss of SUMO-conjugated TFAP2A. The effect of SUMO inhibition on CD44 expression was dependent upon TFAP2A. Treatment with SUMO inhibitors resulted in a statistically improved tumor-free survival in mice harboring 8505C xenografts. An examination of primary ATC tissue determined that TFAP2A was expressed in 4 of 11 tumors surveyed. We conclude that inhibition of the SUMO pathway repressed the CSC population, delaying the outgrowth of tumor xenografts in ATC. The effect of SUMO inhibition was dependent upon expression of SUMO-conjugated TFAP2A, which may serve as a molecular marker for therapeutic effects of SUMO inhibitors. The findings provide pre-clinical evidence for development of SUMO inhibitors for the treatment of ATC.

Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells.

Organization and Regulation of Soybean SUMOylation System under Abiotic Stress Conditions.

Covalent attachment of the small ubiquitin-related modifier, SUMO, to substrate proteins plays a significant role in plants under stress conditions, which can alter target proteins' function, location, and protein-protein interactions. Despite this importance, information about SUMOylation in the major legume crop, soybean, remains obscure. In this study, we performed a bioinformatics analysis of the entire soybean genome and identified 40 genes belonged to six families involved in a cascade of enzymatic reactions in soybean SUMOylation system. The cis-acting elements analysis revealed that promoters of SUMO pathway genes contained different combinations of stress and development-related cis-regulatory elements. RNA-seq data analysis showed that SUMO pathway components exhibited versatile tissue-specific expression patterns, indicating coordinated functioning during plant growth and development. qRT-PCR analysis of 13 SUMO pathway members indicated that majority of the SUMO pathway members were transcriptionally up-regulated by NaCl, heat and ABA stimuli during the 24 h period of treatment. Furthermore, SUMOylation dynamics in soybean roots under abiotic stress treatment were analyzed by western blot, which were characterized by regulation of SUMOylated proteins. Collectively, this study defined the organization of the soybean SUMOylation system and implied an essential function for SUMOylation in soybean abiotic stress responses.

Abstract 1147: Probing the roles of SUMOylation in cancer cell biology using a selective SUMO activating enzyme inhibitor

Abstract SUMOylation has been implicated in many cellular processes that are important for cancer cell survival, including cell cycle, chromosome structure and segregation, nuclear and subnuclear organization, transcription and DNA damage repair. However, a potent and selective inhibitor to target the SUMO pathway has been lacking. The SUMO-activating enzyme (SAE) is an essential enzyme in the pathway that initiates the SUMOylation process. Here we report the identification of the first mechanism-based SAE inhibitors with nanomolar potency in cellular assays. These inhibitors selectively block SAE enzyme activity and total SUMOylation in cells, which leads to reduced cancer cell proliferation. Moreover, SAE inhibition resulted in disruption of PML nuclear bodies and redistribution of DAXX. In vivo administration of SAE inhibitor into tumor bearing mice results in modulation of several biomarkers including a significant reduction in SUMO-conjugates and E2 Ubc9 thioesters, demonstrating SUMO pathway inhibition. Our results demonstrate the feasibility of inhibiting the SUMO pathway with small molecule inhibitors and provide tools to study the SUMO biology in cancer. Citation Format: Sai M Pulukuri, Xingyue He, Stephen Grossman, Jessica Riceberg, Erik Koenig, Teresa Soucy, Keli Song, Anna Kreshock, Dylan England, Hirotake Mizutani, Larry Dick, James Brownell, John Newcomb, Steve Langston, Eric Lightcap, Katherine Galvin. Probing the roles of SUMOylation in cancer cell biology using a selective SUMO activating enzyme inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1147. doi:10.1158/1538-7445.AM2017-1147

SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing.

Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine. Previous work from our laboratory revealed the splicing factor SRSF1 as a regulator of the SUMO pathway, leading us to explore a connection between this pathway and the splicing machinery. We show here that addition of a recombinant SUMO-protease decreases the efficiency of pre-mRNA splicing in vitro. By mass spectrometry analysis of anti-SUMO immunoprecipitated proteins obtained from purified splicing complexes formed along the splicing reaction, we identified spliceosome-associated SUMO substrates. After corroborating SUMOylation of Prp3 in cultured cells, we defined Lys 289 and Lys 559 as bona fide SUMO attachment sites within this spliceosomal protein. We further demonstrated that a Prp3 SUMOylation-deficient mutant while still capable of interacting with U4/U6 snRNP components, is unable to co-precipitate U2 and U5 snRNA and the spliceosomal proteins U2-SF3a120 and U5-Snu114. This SUMOylation-deficient mutant fails to restore the splicing of different pre-mRNAs to the levels achieved by the wild type protein, when transfected into Prp3-depleted cultured cells. This mutant also shows a diminished recruitment to active spliceosomes, compared to the wild type protein. These findings indicate that SUMO conjugation plays a role during the splicing process and suggest the involvement of Prp3 SUMOylation in U4/U6•U5 tri-snRNP formation and/or recruitment.

Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism.

Ubiquitin-like proteins (Ubl’s) are conjugated to target proteins or lipids to regulate their activity, stability, subcellular localization, or macromolecular interactions. Similar to ubiquitin, conjugation is achieved through a cascade of activities that are catalyzed by E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. In this review, we will summarize structural and mechanistic details of enzymes and protein cofactors that participate in Ubl conjugation cascades. Precisely, we will focus on conjugation machinery in the SUMO, NEDD8, ATG8, ATG12, URM1, UFM1, FAT10, and ISG15 pathways while referring to the ubiquitin pathway to highlight common or contrasting themes. We will also review various strategies used to trap intermediates during Ubl activation and conjugation.

Abstract P3-03-13: Chronic inhibition of signal transducer and activator of transcription 3/5 in treatment-resistant human breast cancer cell subtypes: Convergence on the reactive oxyten species/SUMOylation pathway and its effects on xCT expression and system xc- activity

Abstract Pharmacologically targeting activated signal transducer and activator of transcription 3 (STAT3) and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter system xc- contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in aggressive breast cancer cells. We have previously shown that blocking STAT3/5 using the novel small molecule inhibitor SH-4-54, designed to target the SH2 domains of both proteins, increases expression of xCT, which encodes the active component of system xc-, thereby increasing antiporter activity in human breast cancer cells. The current investigation demonstrates that chronic treatment with SH-4-54, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in the triple-negative MDA-MB-231 SH-4-54-resistant clones, with the inverse occurring in estrogen-responsive T47D cells. This “ying-yang” effect is linked with a shifted balance between phosphorylated STAT3 and STAT5, intracellular levels of reactive oxygen species (ROS), STAT5 SUMOylation/de-SUMOylation, and the expression of STAT3/5 target genes (assessed by NextGeneration RNA sequencing). STAT5 emerged as a definitive negative transcriptional regulator of xCT, while STAT3 activation was coupled with increased system xc- activity. Specifically, inhibiting constitutive STAT3 phosphorylation in MDA-MB-231 cells induces ROS, thereby affecting the SUMO pathway by favoring de-SUMOylation and STAT5 phosphorylation. Activated STAT5 is then able to serve as a repressor by binding to its recognition sequence within the xCT promoter, reducing xCT expression and thereby destabilizing an important redox balancing mechanism by limiting cystine uptake through system xc-. In contrast, in ERα-positive cells that initially respond to STAT5-mediated signaling, STAT3 becomes activated and xCT expression is up-regulated in response to chronic SH-4-54 treatment, potentially leading to a more aggressive cancer subtype. Further destabilizing the cellular redox status may therefore be critical to produce clinically meaningful outcomes linked to chronic treatment with potent STAT3/5 inhibitors like SH-4-54. We assessed this notion by treating SH-4-54-resistant MDA-MB-231 clones with specific ROS-inducing reagents, including capsazepine, bleomycin, and paclitaxel, which produced different effects on cell viability. We propose that careful classification of a patient's breast cancer subtype is central to therapeutically targeting STAT3/5 as a means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers. Citation Format: Linher-Melville K, Nashed MG, Ungard R, Haftchenary S, Gunning PT, Singh G. Chronic inhibition of signal transducer and activator of transcription 3/5 in treatment-resistant human breast cancer cell subtypes: Convergence on the reactive oxyten species/SUMOylation pathway and its effects on xCT expression and system xc- activity [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-13.

Manipulating PML SUMOylation via Silencing UBC9 and RNF4 Regulates Cardiac Fibrosis

The promyelocytic leukemia protein (PML) is essential in the assembly of dynamic subnuclear structures called PML nuclear bodies (PML-NBs), which are involved in regulating diverse cellular functions. However, the possibility of PML being involved in cardiac disease has not been examined. In mice undergoing transverse aortic constriction (TAC) and arsenic trioxide (ATO) injection, transforming growth factor β1 (TGF-β1) was upregulated along with dynamic alteration of PML SUMOylation. In cultured neonatal mouse cardiac fibroblasts (NMCFs), ATO, angiotensin II (Ang II), and fetal bovine serum (FBS) significantly triggered PML SUMOylation and the assembly of PML-NBs. Inhibition of SUMOylated PML by silencing UBC9, the unique SUMO E2-conjugating enzyme, reduced the development of cardiac fibrosis and partially improved cardiac function in TAC mice. In contrast, enhancing SUMOylated PML accumulation, by silencing RNF4, a poly-SUMO-specific E3 ubiquitin ligase, accelerated the induction of cardiac fibrosis and promoted cardiac function injury. PML colocalized with Pin1 (a positive regulator for TGF-β1 mRNA expression in PML-NBs) and increased TGF-β1 activity. These findings suggest that the UBC9/PML/RNF4 axis plays a critical role as an important SUMO pathway in cardiac fibrosis. Modulating the protein levels of the pathway provides an attractive therapeutic target for the treatment of cardiac fibrosis and heart failure.

Uncovering the SUMOylation and ubiquitylation crosstalk in human cells using sequential peptide immunopurification

Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine the interrelationship between these modifications. Here, we report an optimized immunoaffinity method that permits the study of both protein ubiquitylation and SUMOylation from a single sample. This method enables the unprecedented identification of 10,388 SUMO sites in HEK293 cells. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification facilitates the dynamic profiling of SUMOylated and ubiquitylated proteins in HEK293 cells treated with the proteasome inhibitor MG132. Quantitative proteomic analyses reveals crosstalk between substrates that control protein degradation, and highlights co-regulation of SUMOylation and ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of proteasome subunits. The SUMOylation of the proteasome affects its recruitment to promyelocytic leukemia protein (PML) nuclear bodies, and PML lacking the SUMO interacting motif fails to colocalize with SUMOylated proteasome further demonstrating that this motif is required for PML catabolism.

The SUMO Pathway in Mitosis.

Mitosis is the stage of the cell cycle during which replicated chromosomes must be precisely divided to allow the formation of two daughter cells possessing equal genetic material. Much of the careful spatial and temporal organization of mitosis is maintained through post-translational modifications, such as phosphorylation and ubiquitination, of key cellular proteins. Here, we will review evidence that sumoylation, conjugation to the SUMO family of small ubiquitin-like modifiers, also serves essential regulatory roles during mitosis. We will discuss the basic biology of sumoylation, how the SUMO pathway has been implicated in particular mitotic functions, including chromosome condensation, centromere/kinetochore organization and cytokinesis, and what cellular proteins may be the targets underlying these phenomena.