Small Ubiquitin-like Modifier Proteins Research Articles

Characterizing the Conformational Dynamics of Human SUMO2: Insights into its Interaction with Metal Ions and SIMs.

SUMO (Small Ubiquitin-like Modifiers) proteins are involved in a crucial post-translational modification commonly termed as SUMOylation. In this work, we have investigated the native-state conformational flexibility of human SUMO2 and its interaction with Cu2+ and Zn2+ ions using 15N-1H based 2D NMR spectroscopy. After SUMO1, SUMO2 is the most studied SUMO isoform in humans which shares 45 % and ~80 % similarity with SUMO1 in terms of sequence and structure, respectively. In this manuscript, we demonstrate that compared to SUMO1, several amino acids around the α1-helix region of SUMO2 access energetically similar near-native conformations. These conformations could play a crucial role in SUMO2's non-covalent interactions with SUMO interaction motifs (SIMs) on other proteins. The C-terminal of SUMO2 was found to bind strongly with Cu2+ ions resulting in a trimeric structure as observed by gel electrophoresis. This interaction seems to interfere in its non-covalent interaction with a V/I-x-V/I-V/I based SIM in Daxx protein.

Abnormal protein SUMOylation in liver disease: novel target for therapy.

SUMOylation is an important protein post-translational modification (PTM) process, in which the small ubiquitin-like modifier (SUMO) protein covalently binds to the target protein and regulates stability, subcellular localization, and protein-protein interaction of the target protein. Protein SUMOylation exerts crucial regulatory function in the liver, and its abnormalities are associated with various liver-related disease processes. This review focuses on the biological functions of protein SUMOylation in liver-related diseases in recent years, summarizes the molecular mechanisms of SUMOylation in the replication of hepatitis viruses and the occurrence of hepatocellular carcinoma, and discusses the significance of SUMOylation in liver-related disorders, which is essential for understanding liver biological processes and formulating therapeutic strategies.

SUMO-specific proteases: SENPs in oxidative stress-related signaling and diseases.

Oxidative stress is employed to depict a series of responses detrimental to normal cellular functions resulting from an imbalance between intracellular oxidants, mainly reactive oxygen species and antioxidant defenses. Oxidative stress often contributes to the development of various diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases. In this process, the relationship between small ubiquitin-like modifier (SUMO) and oxidative stress has garnered significant attention, with its posttranslational modification (PTM) frequently serving as a marker of oxidative stress status. Sentrin/SUMO-specific proteases (SENPs), affected by alternative splicing, PTMs such as phosphorylation and ubiquitination, and various protein interactions, are crucial molecules in the SUMO process. The human SENP family has six members (SENP1-3, SENP5-7), which are classified into two categories based on sequence similarity, substrate specificity, and subcellular location. They have two core functions in the human body: first, by cleaving the precursor SUMO and exposing the C-terminal glycine, they initiate the SUMO process; second, they can specifically recognize and dissociate SUMO proteins bound to substrates, a process known as deSUMOylation. However, the connection between deSUMOylation and oxidative stress remains a relatively unexplored area despite their strong association with oxidative diseases such as cancer and cardiovascular disease. This article aims to illustrate the significant contribution of SENPs to the oxidative stress pathway through deSUMOylation by reviewing their structure and classification, their roles in oxidative stress, and the changes in their expression and activity in several typical oxidative stress-related diseases.

Unveiling success determinants for AMB-assisted phase expansion of fusion proteins in ARP/wARP

Fusion proteins (FPs) are frequently utilized as a biotechnological tool in the determination of macromolecular structures using X-ray methods. Here, we explore the use of different protein tags in various FP, to obtain initial phases by using them in a partial molecular replacement (MR) and constructing the remaining FP structure with ARP/wARP. Usually, the tag is removed prior to crystallization, however leaving the tag on may facilitate crystal formation, and structural determination by expanding phases from known to unknown segments of the complex. In this study, the Protein Data Bank was mined for an up-to-date list of FPs with the most used protein tags, Maltose Binding Protein (MBP), Green Fluorescent Protein (GFP), Thioredoxin (TRX), Glutathione transferase (GST) and the Small Ubiquitin-like Modifier Protein (SUMO). Partial MR using the protein tag, followed by automatic model building, was tested on a subset of 116 FP. The efficiency of this method was analyzed and factors that influence the coordinate construction of a substantial portions of the fused protein were identified. Using MBP, GFP, and SUMO as phase generators it was possible to build at least 75 % of the protein of interest in 36 of the 116 cases tested. Our results reveal that tag selection has a significant impact; tags with greater structural stability, such as GFP, increase the success rate. Further statistical analysis identifies that resolution, Wilson B factor, solvent percentage, completeness, multiplicity, protein tag percentage in the FP (considering amino acids), and the linker length play pivotal roles using our approach. In cases where a structural homologous is absent, this method merits inclusion in the toolkit of protein crystallographers.

Abstract 5393: Characterization of small ubiquitin-like modifier (SUMO) protein complex expression in human cancer cells to identify novel therapeutic targets

Abstract SUMOylation is a post-translational modification where a number of small ubiquitin-like modifier proteins (SUMO) are conjugated to lysine residues of the target proteins and contribute to various cellular functions essential for cancer cell survival and proliferation. The unique behaviors, phenotypic and molecular adaptations found in the naked-mole rat (NMR) suggest a high stability and effective functioning of the molecular machinery that counteracts damage accumulation in its genome. NMR can reach 32 years, shows no signs of ageing, and possesses a very efficient mechanism of resistance to cancer. This study aims to investigate the regulation of SUMOylation machinery between NMR tissues in comparison to human cancer cells, to find novel targets for cancer therapy. Gene expression of SUMO isoforms between NMR and humans, were investigated through qRT-PCR. The data suggests a significant low expression level for most SUMO machinery (except PIAS4) in NMR brain and intestine tissues compared to MCF-7, DLD-1, SH-SY5Y, and MCF-10A cells. Western blotting analysis also revealed high protein expression of PIAS4 in NMR compared to SENP1 and Ubc9. Also, SUMO machinery expression levels from NMR are recapitulated in cancer cells either silencing (hUBE2I: shRNA, hSENP1: shRNA) or overexpression (Myc/hPIAS4: Exp). Further, cytotoxicity assay was analyzed in relevance to Doxorubicin in MCF-7, 5-Fluorouracil in DLD-1 and Methotrexate in SH-SY5. The functional assays such as clonogenic and invasion assays were performed including apoptotic mechanisms (Bcl-2, and BID). In conclusion, the data revealed important differences in SUMOylation machinery between NMR and human cancer cells, which provides a future therapeutic path for cancer treatment. Citation Format: Mohammed Abdullah Mohammed Salih, Ritchie Williamson, Sriharsha Kantamneni. Characterization of small ubiquitin-like modifier (SUMO) protein complex expression in human cancer cells to identify novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5393.

Emerging role of SENP1 in tumorigenesis and cancer therapy.

Acting as a cysteine protease, small ubiquitin-like modifier (SUMO)/sentrin-specific protease1 (SENP1) involved in multiple physiological and pathological processes through processing the precursor SUMO protein into mature form and deSUMOylating target protein. It has been reported that SENP1 is highly expressed and plays a carcinogenic role in various cancers. In this paper, we mainly explore the function and mechanism of SENP1 in tumor cell proliferation, apoptosis, invasion, metastasis, stemness, angiogenesis, metabolism and drug resistance. Furthermore, the research progress of SENP1 inhibitors for cancer treatment is introduced. This study aims to provide theoretical references for cancer therapy by targeting SENP1.

SUMOylation impact on synaptotoxicity of tau oligomers

AbstractBackgroundTau oligomers (oTau) secreted by neurons and astrocytes are linked to the propagation of pathology and negatively affect neuronal activity and viability. oTau‐mediated synaptic loss leads to cognitive deficits and manifests the clinical outcomes of Alzheimer’s disease and related tauopathies. Small Ubiquitin‐like MOdifier (SUMO) proteins regulate multiple cellular events and SUMOylation plays pivotal roles in synaptic biology. Changes in SUMO conjugation and function are also linked to AD and related neurodegenerative disorders such as Huntington’s and Parkinson’s disease. The goal of this investigation is to determine the impact of the two main SUMO isoforms, SUMO1 and SUMO2, on oTau related synaptotoxicity.MethodSUMO1 and SUMO2 transgenic mice were generated with expression regulated by the neuron‐specific prion cos‐tet promoter. These in vivo models were used to generate double transgenic mice expressing human SUMO proteins and P301S mutant Tau. Changes in synaptic density and activity and the effects of oTau pathology were investigated with respect to cognitive deficits and long term potentiation (LTP).ResultElevated expression of human SUMO1 lead to impaired synaptic development and increased tau aggregation. SUMO1 transgenics crossed to Tau mutant mice resulted in an accelerated synaptic loss and a more severe disease phenotype as evidenced by LTP impairments and rapid cognitive decline. Conversely, SUMO2 conjugation levels were decreased in the presence of oTau and phospho‐tau pathology indicating a down‐regulation of its function. Enhanced SUMO2 expression in a mouse model of Tau pathology reversed this process and resulted in a significant reduction in oTau‐mediated synaptotoxicity and the associated LTP and cognitive impairments.ConclusionCumulatively, our findings indicate that SUMO1 negative impacts and exacerbates oTau‐related synaptic dysfunction. In contrast, SUMO2 confers substantial neuroprotection and represents a potential therapeutic avenue to counteract oTau‐induced synaptotoxicity in AD.

Targeting Sumoylation Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment

Acute myeloid leukemia (AML) is a hematopoietic cancer characterized by aberrant immature myeloid progenitor blasts in bone marrow and peripheral blood. Venetoclax (VEN), a BCL-2 inhibitor, is FDA-approved for AML treatment with hypomethylating agents (HMA). Despite the improvement of VEN and HMA, not all patients respond, and most patients develop resistance. This presents an urgent need for new therapies to overcome VEN resistance and enhance VEN efficacy. We propose SUMOylation inhibition as a novel therapy to address this need. SUMOylation regulates protein function by covalently attaching Small Ubiquitin-like MOdifier (SUMO) proteins to target proteins. SUMOylation plays an important role in tumor progression and immune response. Our study aims to evaluate the effects of SUMOylation inhibition on anti-AML activity of VEN and the mechanism. We firstly generated VEN-resistant cell lines Molm13-VR and MV411-VR by culturing Molm13 and MV4-11 cells in increasing doses of VEN. SUMO E1 (SAE2) and global SUMOylation levels were remarkably upregulated in VEN-resistant cell lines, suggesting SUMOylation plays a role in VEN resistance. We evaluated the antileukemic effects of TAK-981 (subasumstat), a novel and specific SUMO E1 inhibitor, alone and in combination with VEN, using AML cell lines and primary blasts isolated from refractory/relapsed (R/R) AML patients. Different AML cell lines and blast samples showed various sensitivities to VEN (IC 50 10nM ~ 5,000nM), but significant synergism was observed with TAK-981 and VEN in cell viability assay. TAK-981 synergized with VEN at inducing AML cell apoptosis determined by annexin-V staining with flow cytometry as well as increased levels of cleaved-PARP and cleaved caspase-3 by western blotting. The mechanism of action of VEN is dependent on BCL-2 mediated mitochondrial function and metabolism. We assessed mitochondrial membrane potential using TMRM probe. TAK-981 reduced mitochondrial membrane potential in both VEN-sensitive and resistant AML lines, further in combination with VEN. Because mitochondrial membrane potential is maintained by normal mitochondrial structure, we performed electron microscopy to determine mitochondrial structure. VEN had limited effect on mitochondria in resistant cells. However, TAK-981 induced abnormal ultrastructure mitochondria with fewer cristae, worsened by TAK-981+VEN combination. Since TAK-981 treatment caused mitochondrial structure defects, we determine the mitochondrial function using Seahorse XF assay. VEN decreased oxygen consumption rate (OCR), indicating inhibition of oxidative phosphorylation (OXPHOS), further suppressed by TAK-981. Metabolomic profiling by mass spectrometry indicated TAK-981+VEN treated AML cells displayed striking decreases in TCA cycle intermediates and reduced electron transport chain (ETC) activity. Pathway analysis of the metabolomics data revealed significant enrichment in amino acids metabolism. Notably, RNA-sequencing and GSEA analysis showed consistent findings that TAK-981+VEN showed suppressed signaling pathways in mitochondrial function, glycolysis and amino-acid metabolisms, indicating TAK-981 targeted mitochondrial metabolism to enhance VEN activity. We then determined whether TAK-981 enhanced antileukemic effect of VEN ex vivo and in vivo. TAK-981 synergized with VEN at reducing colony formation capacity in colony formation assay using leukemia stem cells (LSCs) isolated from relapse AML blasts. More importantly, TAK-981 showed remarkable therapeutic effect in patient-derived xenograft (PDX) mouse model engrafted with blasts isolated from R/R AML patients, and significantly synergized with VEN at extending mice survival in vivo. In conclusion, TAK-981 enhances the antileukemic activity of VEN by targeting mitochondria function and metabolism, offering a potent therapy for refractory/relapsed AML.

Metabolic Reprograming of Exhausted Intratumoral CD8+ T-Cell Underlies Anti-Tumor Activity of Sumoylation Inhibitors in Large B Cell Lymphoma

Immune checkpoint inhibitors (ICI) targeting PD1 have revolutionized our approach to solid tumors and Hodgkin lymphoma, but they have not been effective in treating Large B Cell Lymphoma (LBCL). In prior work, we used single cell spatial profiling of human tumors to show that exhausted CD8 tumor and TREG populations were associated with PD1 resistance in LBCL (Colombo et al. Blood Adv 2022). Here we show, using a syngeneic model of LBCL, that PD1 resistance is associated with lower CD8+ T-cell basal metabolic rate (BMR) in tumor infiltrating lymphocytes (TIL), and that treatment with the covalent SUMOylation inhibitor (SB-4826) increases the metabolic rate of TIL by metabolic reprograming. Single cell metabolic profiling of human LBCL is on-going and will be presented as well. In summary these studies demonstrate that metabolic dysfunction of T cells in LBCL underlie resistance to PD1 checkpoint inhibitors and that this can be overcome by treatment with SUMOlyation inhibitors. Direct measurement of intra-tumoral metabolic state is difficult to perform on clinical samples, however large tumor size is hypothesized to be a surrogate for increased metabolic derangement. As tumors expand and become more metabolically active themselves, infiltrating immune cells experience decreased O2 tension as well as nutrient deprivation, ultimately leading to decreased anti-tumor immune responses. In support of this idea, it has been observed that high metabolic tumor volume at baseline predicts survival in LBCL patients, independent of their initial response to therapy. We have shown that mice with established (>125mm 3) A20 tumors are resistant to anti-PD1 therapy. However, A20 bearing mice treated ~5 days post implantation are responsive to anti-PD1 therapy. To understand these findings, we examined sensitive (small) and resistant (large) A20 tumors by flow cytometry analysis and found that resistant tumors had lower relative numbers of T cells, lower absolute numbers of CD8+ T cells, increased absolute numbers of TREG and a strikingly decreased CD8/TREG ratio. Additionally, the resistant tumors had increased absolute numbers of pre-exhausted and exhausted CD8+ T cells as well as decreased BMR. Small ubiquitin-like modifiers (SUMO) proteins regulate a variety of cellular processes in tumor and immune cells and inhibitors of SUMOylation have shown anti-tumor activity in a variety of preclinical tumor models with potential synergy with checkpoint inhibitors targeting PD-1 (Cannon et al. Cancer Res, 2023). We demonstrate that a dose of 100mg/kg of SB-4826 led to a complete response in all mice treated (Fig. 1A) and we also show that this effect is dependent on CD8+ T-cells and elicits a long term memory response, with 0 of 15 mice in remission developing tumor upon rechallenge. A low dose (25mg/kg) of SB-4826 failed to elicit a response in any of the mice treated while anti-PD1 therapy elicited a response in 1/6 mice (Fig. 1A). Combination of 25mg/kg SB-4826 with anti-PD1 therapy demonstrated synergism with a response in 3/6 mice (Fig. 1A). We also demonstrate that global SUMOylation levels are higher in PD1+ TIM3+ CD8+ TILs compared to PD1- TIM3- CD8+ T-cells, suggesting that SUMOylation may be acting as a negative regulator of T-cell function. Using SCENITH (Arguello et al. Cell Metabolism 2020) for single cell metabolic studies, we demonstrate that SB-4826 increases the BMR (Fig. 1B), mitochondrial dependency and proliferative capacity of tumor infiltrating PD1+ TIM3+ CD8+ T-cells (TILs). While both SB-4826 and PD1 inhibition led to increased CD8+ TIL numbers, SB-4826 led to increased BMR in CD8+ TILs while PD1 inhibition did not affect CD8+ TILs BMR. Additionally, PD1 inhibition significantly decreased CD8+ TILs mitochondrial dependency for metabolism, while SB-4826 increased mitochondrial dependency (Fig. 1B). Additionally, we recapitulate these metabolic enhancements in an in vitro system of T-cell activation, further confirming a T-cell intrinsic effect following SB-4826. Mechanistically, we found that SB-4826 led to increased HIF-1a levels in both CD4+ and CD8+ cells following TCR activation, likely through stabilization of HIF-1a due to decreased SUMOylation-dependent turnover (Cheng J et al. Cell 2007). We hypothesize that metabolic reprograming of CD8 T-cells through increased HIF-1a is the basis for synergy between SUMOylation inhibition and immune checkpoint inhibitors in LBCL.

Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

SENP1 knockdown-mediated CTCF SUMOylation enhanced its stability and alleviated lipopolysaccharide-evoked inflammatory injury in human lung fibroblasts via regulation of FOXA2 transcription

BackgroundExcessive inflammation is the main cause of treatment failure in neonatal pneumonia (NP). CCCTC-binding factor (CTCF) represents an important node in various inflammatory diseases. In the present study, we tried to clarify the function and underlying molecular mechanism of CTCF on an in vitro cellular model of NP, which was generated by simulating the human lung fibroblast cell line WI-38 with lipopolysaccharide (LPS). MethodsThe SUMOylation level and protein interaction were verified by Co-immunoprecipitation assay. Cell viability was measured by Cell Counting Kit-8 assay. Inflammatory factors were examined by Enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by TUNEL assay. The binding activity of CTCF to target promoter was tested by chromatin immunoprecipitation and luciferase reporter assay. ResultsLPS treatment restrained cell viability, promoted the production of inflammatory factors, and enhanced cell apoptosis. CTCF overexpression played anti-inflammatory and anti-apoptotic roles. Furthermore, CTCF was modified by SUMOylation with small ubiquitin-like modifier protein 1 (SUMO1). Interfering with sumo-specific protease 1 (SENP1) facilitated CTCF SUMOylation and protein stability, thus suppressing LPS-evoked inflammatory and apoptotic injuries. Moreover, CTCF could bind to the forkhead box protein A2 (FOXA2) promoter region to promote FOXA2 expression. The anti-inflammatory and anti-apoptotic roles of CTCF are associated with FOXA2 activation. In addition, SENP1 knockdown increased FOXA2 expression by enhancing the abundance and binding ability of CTCF. ConclusionsSUMOylation of CTCF by SENP1 knockdown enhanced its protein stability and binding ability and it further alleviated LPS-evoked inflammatory injury in human lung fibroblasts by positively regulating FOXA2 transcription.

SUMOylation Modulates Reactive Oxygen Species (ROS) Levels and Acts as a Protective Mechanism in the Type 2 Model of Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is the prevalent type of peripheral neuropathy; it primarily impacts extremity nerves. Its multifaceted nature makes the molecular mechanisms of diabetic neuropathy intricate and incompletely elucidated. Several types of post-translational modifications (PTMs) have been implicated in the development and progression of DPN, including phosphorylation, glycation, acetylation and SUMOylation. SUMOylation involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, and it plays a role in various cellular processes, including protein localization, stability, and function. While the specific relationship between high blood glucose and SUMOylation is not extensively studied, recent evidence implies its involvement in the development of DPN in type 1 diabetes. In this study, we investigated the impact of SUMOylation on the onset and progression of DPN in a type 2 diabetes model using genetically modified mutant mice lacking SUMOylation, specifically in peripheral sensory neurons (SNS-Ubc9-/-). Behavioural measurement for evoked pain, morphological analyses of nerve fibre loss in the epidermis, measurement of reactive oxygen species (ROS) levels, and antioxidant molecules were analysed over several months in SUMOylation-deficient and control mice. Our longitudinal analysis at 30 weeks post-high-fat diet revealed that SNS-Ubc9-/- mice exhibited earlier and more pronounced thermal and mechanical sensation loss and accelerated intraepidermal nerve fibre loss compared to control mice. Mechanistically, these changes are associated with increased levels of ROS both in sensory neuronal soma and in peripheral axonal nerve endings in SNS-Ubc9-/- mice. In addition, we observed compromised detoxifying potential, impaired respiratory chain complexes, and reduced levels of protective lipids in sensory neurons upon deletion of SUMOylation in diabetic mice. Importantly, we also identified mitochondrial malate dehydrogenase (MDH2) as a SUMOylation target, the activity of which is negatively regulated by SUMOylation. Our results indicate that SUMOylation is an essential neuroprotective mechanism in sensory neurons in type 2 diabetes, the deletion of which causes oxidative stress and an impaired respiratory chain, resulting in energy depletion and subsequent damage to sensory neurons.

UFMylation of HRD1 regulates endoplasmic reticulum homeostasis.

Ubiquitin fold modifier 1 is a small ubiquitin-like protein modifier that is essential for embryonic development of metazoans. Although UFMylation has been connected to endoplasmic reticulum homeostasis, the underlying mechanisms and the relevant cellular targets are largely unknown. Here, we show that HRD1, a ubiquitin ligase of ER-associated protein degradation (ERAD), is a novel substrate of UFM1 conjugation. HRD1 interacts with UFMylation components UFL1 and DDRGK1 and is UFMylated at Lys610 residue. In UFL1-depleted cells, the stability of HRD1 is increased and its ubiquitination modification is reduced. In the event of ER stress, the UFMylation and ubiquitination modification of HRD1 is gradually inhibited over time. Alteration of HRD1 Lys610 residue to arginine impairs its ability to degrade unfolded or misfolded proteins to disturb protein processing in ER. These results suggest that UFMylation of HRD1 facilitates ERAD function to maintain ER homeostasis.

Inhibition of SUMOylation promotes remyelination and reduces IL-17 mediated autoimmune inflammation: Novel approach toward treatment of inflammatory CNS demyelinating disease

Small ubiquitin like modifiers (SUMO) are reversible posttranslational modifiers of intracellular proteins. In the CNS, expression of myelin genes is regulated by state of SUMOylation of their respective transcription factors. In the immune system, deSUMOylation activates innate immune responses and promotes anti-viral immunity. However, the role played by SUMO in an adaptive immune response and in the development of T cell mediated autoimmune disease has not been previously described.TAK981 is a synthetic small molecule which by forming adducts with SUMO proteins prevents SUMOylation. We examined the expression of myelin genes and their transcription factors following culture with TAK981 in Oligodendrocyte Precursor Cells (OPC). We found that myelin basic protein (MBP), a key myelin protein, is upregulated in OPC in the presence of TAK981. We also found increased expression of transcription factors Sox10 and Myrf, which engage in the expression of MBP. In the Cuprizone model of demyelination/remyelination, animals which were treated with TAK981 showed increased remyelination in areas of demyelination and an increase in the number of maturing oligodendrocytes compared to vehicle treated controls. In in vitro cultures of lymphocytes, TAK981 reduced the expression of TH17 in T cells in mice immunized with MOGp35–55. Following in vivo treatment with TAK981, there was a significant reduction in the clinical and pathological severity in mice immunized to develop experimental allergic encephalitis (EAE).The dual effects of deSUMOylation on remyelination and in regulating an autoimmune adaptive response offers a novel approach to the management of human inflammatory demyelinating diseases such as multiple sclerosis.

SUMOylation indirectly suppresses activity of the HIF-1α pathway in intestinal epithelial cells

The hypoxia-inducible factor (HIF) is a master regulator of the cellular transcriptional response to hypoxia. While the oxygen-sensitive regulation of HIF-1α subunit stability via the ubiquitin-proteasome pathway has been well described, less is known about how other oxygen-independent post-translational modifications impact the HIF pathway. SUMOylation, the attachment of SUMO (small ubiquitin-like modifier) proteins to a target protein, regulates the HIF pathway, although the impact of SUMO on HIF activity remains controversial. Here, we examined the effects of SUMOylation on the expression pattern of HIF-1α in response to pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) in intestinal epithelial cells. We evaluated the effects of SUMO-1, SUMO-2, and SUMO-3 overexpression and inhibition of SUMOylation using a novel selective inhibitor of the SUMO pathway, TAK-981, on the sensitivity of HIF-1α in Caco-2 intestinal epithelial cells. Our findings demonstrate that treatment with TAK-981 decreases global SUMO-1 and SUMO-2/3 modification and enhances HIF-1α protein levels, whereas SUMO-1 and SUMO-2/3 overexpression results in decreased HIF-1α protein levels in response to DMOG. Reporter assay analysis demonstrates reduced HIF-1α transcriptional activity in cells overexpressing SUMO-1 and SUMO-2/3, whereas pretreatment with TAK-981 increased HIF-1α transcriptional activity in response to DMOG. In addition, HIF-1α nuclear accumulation was decreased in cells overexpressing SUMO-1. Importantly, we showed that HIF-1α is not directly SUMOylated, but that SUMOylation affects HIF-1α stability and activity indirectly. Taken together, our results indicate that SUMOylation indirectly suppresses HIF-1α protein stability, transcriptional activity, and nuclear accumulation in intestinal epithelial cells.

Exome sequencing links the SUMO protease SENP7 with fatal arthrogryposis multiplex congenita, early respiratory failure and neutropenia

BackgroundSUMOylation involves the attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues on thousands of substrates with target-specific effects on protein function. Sentrin-specific proteases (SENPs) are proteins involved...

Assessment of the Cardiac Noncoding Transcriptome by Single-Cell RNA Sequencing Identifies FIXER, a Conserved Profibrogenic Long Noncoding RNA.

Cardiac fibroblasts have crucial roles in the heart. In particular, fibroblasts differentiate into myofibroblasts in the damaged myocardium, contributing to scar formation and interstitial fibrosis. Fibrosis is associated with heart dysfunction and failure. Myofibroblasts therefore represent attractive therapeutic targets. However, the lack of myofibroblast-specific markers has precluded the development of targeted therapies. In this context, most of the noncoding genome is transcribed into long noncoding RNAs (lncRNAs). A number of lncRNAs have pivotal functions in the cardiovascular system. lncRNAs are globally more cell-specific than protein-coding genes, supporting their importance as key determinants of cell identity. In this study, we evaluated the value of the lncRNA transcriptome in very deep single-cell RNA sequencing. We profiled the lncRNA transcriptome in cardiac nonmyocyte cells after infarction and probed heterogeneity in the fibroblast and myofibroblast populations. In addition, we searched for subpopulation-specific markers that can constitute novel targets in therapy for heart disease. We demonstrated that cardiac cell identity can be defined by the sole expression of lncRNAs in single-cell experiments. In this analysis, we identified lncRNAs enriched in relevant myofibroblast subpopulations. Selecting 1 candidate we named FIXER (fibrogenic LOX-locus enhancer RNA), we showed that its silencing limits fibrosis and improves heart function after infarction. Mechanitically, FIXER interacts with CBX4, an E3 SUMO protein ligase and transcription factor, guiding CBX4 to the promoter of the transcription factor RUNX1 to control its expression and, consequently, the expression of a fibrogenic gene program.. FIXER is conserved in humans, supporting its translational value. Our results demonstrated that lncRNA expression is sufficient to identify the various cell types composing the mammalian heart. Focusing on cardiac fibroblasts and their derivatives, we identified lncRNAs uniquely expressed in myofibroblasts. In particular, the lncRNA FIXER represents a novel therapeutic target for cardiac fibrosis.

Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins

BackgroundSome peptides are targets for degradation when heterologously expressed as fusion proteins in E. coli, which can limit yields after isolation and purification. We recently reported that peptide degradation may be prevented by production of a “sandwiched” SUMO-peptide-intein (SPI) fusion protein, which protects the target peptide sequence from truncation and improves yield. This initial system required cloning with two commercially available vectors. It used an N-terminal polyhistidine tagged small ubiquitin-like modifier (SUMO) protein and a C-terminal engineered Mycobacterium xenopii DNA Gyrase A intein with an inserted chitin binding domain (CBD) to create “sandwiched” fusion proteins of the form: His6-SUMO-peptide-intein-CBD. However, the major drawback of this previously reported fusion protein “sandwich” approach is the increased time and number of steps required to complete the cloning and isolation procedures, relative to the simple procedures to produce recombinant peptides in E. coli from a single (non-“sandwiched”) fusion protein system.ResultsIn this work we generate the plasmid pSPIH6, which improves upon the previous system by encoding both the SUMO and intein proteins and allows facile construction of a SPI protein in a single cloning step. Additionally, the Mxe GyrA intein encoded in pSPIH6 contains a C-terminal polyhistidine tag, resulting in SPI fusion proteins of the form: His6-SUMO-peptide-intein-CBD-His6. The dual polyhistidine tags greatly simplify isolation procedures compared to the original SPI system, which we have here demonstrated with two linear bacteriocin peptides: leucocin A and lactococcin A. The yields obtained for both peptides after purification were also improved compared to the previous SPI system as a result of this streamlined protocol.ConclusionsThis modified SPI system and its simplified cloning and purification procedures described here may be generally useful as a heterologous E. coli expression system to obtain pure peptides in high yield, especially when degradation of the target peptide is an issue.

Abstract 348: Elucidation of SUMOylation machinery in human cancer cells and naked mole-rat tissues

Abstract Background: SUMOylation is a post-translational modification where small ubiquitin-like modifier (SUMO) proteins are conjugated to lysine residues of target proteins. SUMO proteins exist in all eukaryotes and participate in a catalytic cycle, which includes maturation, activation, conjugation, ligation, and de-modification. SUMOylation is known to regulate numerous molecular regulatory mechanisms such as: chromatin remodeling, DNA repair, immune responses, cell cycle progression, apoptosis, and the intracellular signal transduction. SUMOylation dysregulation is known to promote several diseases, including cancer cell survival. The unique traits of phenotypic and molecular adaptations found in naked mole rats (NMR) suggest high stability and effective functioning of the molecular machinery that counteract damage accumulation in its genome. NMR lifespan can reach up to 32 years and acquires a very efficient mechanism of resistance to cancer. This study aims to investigate the regulation of SUMO machinery between human cancer cells breast (MCF-7), colorectal (DLD-1), and neuroblastoma (SH-SY5Y)) and NMR brain and intestinal tissues, in order to find novel targets for cancer treatment. Results: The pairwise alignment identified the similarity of both SUMO genes and proteins including SUMO1, SUMO2/3, and SUMO components of Sentrin-specific protease isoforms (SENP 1-7), activation (SAE1 and Uba2), conjugation (Ubc9), and ligation isoforms (PIAS1-4) between NMR and human. In addition, qRT-PCR data revealed low expression of SUMO components at the mRNA level in NMR brain and intestinal tissues compared to MCF-7, DLD-1, and SH-SY5Y cell lines. On the contrary, PIAS4 expression is significantly higher in the NMR tissues compared to cancer cell lines. Protein analysis via Western blotting performed on MCF-7, DLD-1, and SH-SY5Y cell lines revealed similar levels to mRNA compared to NMR tissue. Ongoing studies are focusing on several functional assays to investigate effects of hUBE2I, hSENP1, and Myc/hPIAS4 effects on cytotoxicity and signaling properties of the cancer cells using techniques such as Microculture Tetrazolium (MTT), migration, invasion, and colony formation. The effect on cytotoxicity is also analyzed in relevance to clinical used treatments such as Doxorubicin in MCF-7, 5-Fluorouracil for DLD-1 and Methotrexate in SH-SY5Y. Conclusion: Expression analysis of SUMOylation machinery revealed lower expression of mRNA levels for SUMO components was observed in NMR tissues compared to all human cancer cells. However, PIAS4 expression was significantly higher in the NMR tissues compared to cancer cell lines. This data revealed important differences in NMR SUMOylation machinery (such as UBE2I, SENP1 and PIAS4) which provides a future therapeutic avenue to treat cancer. Citation Format: Mohammed Abdullah Mohammed Salih, Ritchie Williamson, Sriharsha Kantamneni. Elucidation of SUMOylation machinery in human cancer cells and naked mole-rat tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 348.

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The cover image is based on the Research Article Post-ovulatory aging is associated with altered patterns for small ubiquitin-like modifier (SUMO) proteins and SUMO-specific proteases by Weber Beringui Feitosa et al., https://doi.org/10.1096/fj.202200622R.