Small Ubiquitin-like Modifier Conjugation Research Articles

Insights into the role of SUMO in regulating drought stress responses in pigeonpea (Cajanus cajan).

We have identified and analyzed 28 SUMO-pathway proteins from pigeonpea. Enhanced transcripts of pathway genes and increased SUMO conjugation under drought signifies the role of SUMO in regulating stress. Being a protein-rich and nutrient-dense legume crop, pigeonpea (Cajanus cajan) holds a vital position in a vegetarian meal. It is a resilient crop capable of striving in harsh climates and provides a means of subsistence to small-holding farmers. Nevertheless, extremes of water scarcity and drought conditions, especially during seedling and reproductive stages, remains a major issue severely impacting the growth and overall productivity of pigeonpea. Small ubiquitin-like modifier (SUMO), a post-translational modification system, plays a pivotal role in fortifying plants against stressful conditions by rapid reprogramming of molecular events. In this study, we have scanned the entire pigeonpea genome and identified 28 candidates corresponding to SUMO machinery components of pigeonpea. qRT-PCR analysis of different SUMO machinery genes validated their presence under natural conditions. The analysis of the promoters of identified SUMO machinery genes revealed the presence of abiotic stress-related cis-regulatory elements highlighting the potential involvement of the genes in abiotic stress responses. The transcript level analysis of selected SUMO machinery genes and global SUMO status of pigeonpea proteins in response to drought stress suggests an integral role of SUMO in regulating drought stress conditions in pigeonpea. Collectively, the work puts forward a detailed in silico analysis of pigeonpea SUMO machinery candidates and highlights the essential role of SUMOylation in drought stress responses. Being the first report on a pulse crop, the study will serve as a resource for devising strategies for counteracting drought stress in pigeonpea that can be further extended to other pulse crops.

Abstract 6605: Understanding the role of sumoylated Etv1 in mammary oncogenesis

Abstract The Small Ubiquitin-like Modifier (SUMO) pathway is a major post-translational regulatory mechanism involved in a variety of cellular functions including the DNA damage response, transcriptional regulation, and carcinogenesis. Recent discoveries by our group demonstrated that inhibiting the SUMO pathway can repress the growth of breast cancer xenografts and Neu-driven mammary oncogenesis in a genetically engineered mouse model with conditional knockout (CKO) of the SUMOylation E2 conjugating enzyme, Ube2i. However, despite the consistency of the findings, there remains considerable debate concerning the mechanisms through which SUMO inhibition represses tumorigenesis. Our downstream analysis suggested ETS Translocation Variant 1, encoded by ETV1 gene, acts as a SUMO-sensitive transcription factor that drives mammary oncogenesis and cancer growth. To examine oncogenic properties of SUMO-conjugated ETV1 in breast cancer, we cloned HA-tagged WT Etv1, SUMO-resistant Etv15xKR, or SUMO mimetic SUMO-1-Etv15xKR into a lentiviral vector with a GFP cassette. Proliferation, colony formation in soft agar, and in vivo xenograft assays were evaluated in the Ube2iF/F cell line derived from a tumor in a MMTV-c-neu/Ube2ifl/fl animal. We did ChIP-seq in Ube2iF/F cells using SUMO-1 and Etv1 antibodies to examine the effects of SUMO conjugation on Etv1 genomic occupancy. RNA-seq using siEtv1 or AdCre was also performed to compare changes in gene expression with SUMO-conjugated Etv1 genomic occupancy. Inhibition of SUMOylation via AdCre caused a significant reduction in proliferation and colony formation in vitro and completely block the formation of flank xenografts in vivo. Interestingly, expressing a SUMO-resistant ETV15xKR rescued in vitro growth effects, whereas a SUMO mimetic SUMO-1-ETV15xKR had no effect on restoring proliferation. On the contrary, in vivo xenograft assays demonstrated that the Etv15xKR-SUMO1 efficiently induced tumor formation despite the loss of Ube2i while the sumo-resistant Etv15xKR was unable to rescue tumorigenesis. Bulk RNA-seq and ChIP seq performed demonstrated different patterns of gene regulation between sumoylated and unsumoylated Etv1. Gene set enrichment analysis (GSEA) showed genes associated with cancer stemness were significantly enriched in cells expressing Etv15xKR-SUMO1. Collectively, these observations support Etv1 acting as a SUMO-sensitive transcription factor that drives a stem cell phenotype necessary for mammary oncogenesis and progression. Citation Format: Zhijie Li, Kelsey E. Koch, Dakota T. Thompson, Dana M. Van der Heide, Jeremy Chang, Christopher M. Franke, Mohammed O. Suraju, Anna C. Beck, Allison W. Lorenzen, Jeffrey R. White, Mikhail V. Kulak, David K. Meyerholz, Colin Kenny, Ronald J. Weigel. Understanding the role of sumoylated Etv1 in mammary oncogenesis [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 6605.

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.

Sumoylation in astrocytes induces changes in the proteome of the derived small extracellular vesicles which change protein synthesis and dendrite morphology in target neurons

Emerging evidence highlights the relevance of the protein post-translational modification by SUMO (Small Ubiquitin-like Modifier) in the central nervous system for modulating cognition and plasticity in health and disease. In these processes, astrocyte-to-neuron crosstalk mediated by extracellular vesicles (EVs) plays a yet poorly understood role. Small EVs (sEVs), including microvesicles and exosomes, contain a molecular cargo of lipids, proteins, and nucleic acids that define their biological effect on target cells. Here, we investigated whether SUMOylation globally impacts the sEV protein cargo. For this, sEVs were isolated from primary cultures of astrocytes by ultracentrifugation or using a commercial sEV isolation kit. SUMO levels were regulated: 1) via plasmids that over-express SUMO, or 2) via experimental conditions that increase SUMOylation, i.e., by using the stress hormone corticosterone, or 3) via the SUMOylation inhibitor 2-D08 (2′,3′,4′-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one). Corticosterone and 2-D08 had opposing effects on the number of sEVs and on their protein cargo. Proteomic analysis showed that increased SUMOylation in corticosterone-treated or plasmid-transfected astrocytes increased the presence of proteins related to cell division, transcription, and protein translation in the derived sEVs. When sEVs derived from corticosterone-treated astrocytes were transferred to neurons to assess their impact on protein synthesis using the fluorescence non-canonical amino acid tagging assay (FUNCAT), we detected an increase in protein synthesis, while sEVs from 2-D08-treated astrocytes had no effect. Our results show that SUMO conjugation plays an important role in the modulation of the proteome of astrocyte-derived sEVs with a potential functional impact on neurons.

SUMO protease SENP6 protects the nucleus from hyperSUMOylation-induced laminopathy-like alterations

The small ubiquitin-like modifier (SUMO) protease SENP6 disassembles SUMO chains from cellular substrate proteins. We use a proteomic method to identify putative SENP6 substrates based on increased apparent molecular weight after SENP6 depletion. Proteins of the lamin family of intermediate filaments show substantially increased SUMO modification after SENP6 depletion. This is accompanied by nuclear structural changes remarkably like those associated with laminopathies. Two SUMO attachment sites on lamin A/C are close to sites of mutations in Emery-Driefuss and limb girdle muscular dystrophy. To establish a direct link between lamin SUMOylation and the observed phenotype, we developed proximity-induced SUMO modification (PISM), which fuses a lamin A/C targeting DARPin to a SUMO E3 ligase domain. This directly targets lamin A/C for SUMO conjugation and demonstrates that enhanced lamin SUMO modification recapitulates the altered nuclear structure manifest after SENP6 depletion. This shows SENP6 activity protects the nucleus against hyperSUMOylation-induced laminopathy-like alterations.

Arabidopsis-expressing lysine-null SUMO1 reveals a non-essential role for secondary SUMO modifications in plants.

The reversible conjugation of small ubiquitin-like modifier (SUMO) to other proteins has pervasive roles in various aspects of plant development and stress defense through its selective attachment to numerous intracellular substrates. An intriguing aspect of SUMO is that it can be further modified by SUMOylation and ubiquitylation, which isopeptide-link either or both polypeptides to internal lysines within previously bound SUMOs. Although detectable by mass spectrometry, the functions of these secondary modifications remain obscure. Here, we generated transgenic Arabidopsis that replaced the two related and essential SUMO isoforms (SUMO1 and SUMO2) with a lysine-null SUMO1 variant (K0) immune to further SUMOylation/ubiquitylation at these residues. Remarkably, homozygous SUMO1(K0) sumo1 sumo2 plants developed normally, were not hypersensitive to heat stress, and have nearly unaltered SUMOylation profiles during heat shock. However, subtle changes in tolerance to salt, paraquat, and the DNA-damaging agents bleomycin and methane methylsulfonate were evident, as were increased sensitivities to ABA and the gibberellic acid biosynthesis inhibitor paclobutrazol, suggesting roles for these secondary modifications in stress defense, DNA repair, and hormone signaling. We also generated viable sumo1 sumo2 lines expressing a SUMO1(K0) variant specifically designed to help isolate SUMO conjugates and map SUMOylation sites, thus offering a new tool for investigating SUMO in planta.

MAVS deSUMOylation by SENP1 inhibits its aggregation and antagonizes IRF3 activation.

Mitochondrial antiviral signaling protein (MAVS) is an adapter that recruits and activates IRF3. However, the mechanisms underpinning the interplay between MAVS and IRF3 are largely unknown. Here we show that small ubiquitin-like modifier (SUMO)-specific protease 1 negatively regulates antiviral immunity by deSUMOylating MAVS. Upon virus infection, PIAS3-induced poly-SUMOylation promotes lysine 63-linked poly-ubiquitination and aggregation of MAVS. Notably, we observe that SUMO conjugation is required for MAVS to efficiently produce phase-separated droplets through association with a newly identified SUMO-interacting motif (SIM) in MAVS. We further identify a yet-unknown SIM in IRF3 that mediates its enrichment to the multivalent MAVS droplets. Conversely, IRF3 phosphorylation at crucial residues close to SIM rapidly disables SUMO-SIM interactions and releases activated IRF3 from MAVS. Our findings implicate SUMOylation in MAVS phase separation and suggest a thus far unknown regulatory process by which IRF3 can be efficiently recruited and released to facilitate timely activation of antiviral responses.

Rice E3 Ligase-Like Protein OsPIAL1 Positively Regulated the Drought Stress Response but Negatively Regulated the Salt Stress Response

Small ubiquitin-like modifier (SUMO) E3 ligases that facilitate the conjugation of SUMO proteins to target substrates contain an SP-RING domain which is like the RING domain found in ubiquitin E3 ligases. In this study, we isolated and characterized the <i>Oryza sativa</i> protein inhibitor of activated STAT like1 (OsPIAL1) containing SP-RING domains, as the rice homolog of <i>Arabidopsis</i> PIALs. OsPIAL1 interacts with OsSUMO proteins but does not interact with rice SUMO-conjugating enzymes (OsSCEs). An analysis of transgenic rice plant shows that OsPIAL1 is involved in SUMO conjugation to SCEs but not in SUMO conjugation to substrates. In addition, this OsPIAL1 activity requires drought stress conditions. Expression profiles show that the <i>OsPIAL1</i> gene is induced by only drought stress in the leaves, whereas it is repressed by ABA and abiotic stresses in the roots. Salt stress leads to the fastest decrease in <i>OsPIAL1</i> transcripts in the roots. Furthermore, the stress experiments indicate that the transgenic rice plants overexpressing <i>OsPIAL1</i> exhibit a drought stress-tolerant phenotype but a salt stress hypersensitive phenotype. Our results and those from <i>Arabidopsis pial</i> mutants suggest that PIALs act as a positive regulator in the drought stress response but as a negative regulator in the salt stress response.

Pepper SUMO E3 ligase CaDSIZ1 enhances drought tolerance by stabilizing the transcription factor CaDRHB1.

Small ubiquitin-like modifier (SUMO) conjugation (SUMOylation) is a reversible post-translational modification associated with protein stability and activity, and modulates hormone signaling and stress responses in plants. Previously, we reported that the pepper dehydration-responsive homeobox domain transcription factor CaDRHB1 acts as a positive modulator of drought response. Here, we show that CaDRHB1 protein stability is enhanced by SUMO E3 ligase Capsicum annuum DRHB1-interacting SAP and Miz domain (SIZ1) (CaDSIZ1)-mediated SUMOylation in response to drought, thereby positively modulating abscisic acid (ABA) signaling and drought responses. Substituting lysine (K) 138 of CaDRHB1 with arginine reduced CaDSIZ1-mediated SUMOylation, indicating that K138 is the principal site for SUMO conjugation. Virus-induced silencing of CaDSIZ1 promoted CaDRHB1 degradation, suggesting that CaDSIZ1 is involved in drought-induced SUMOylation of CaDRHB1. CaDSIZ1 interacted with and facilitated SUMO conjugation of CaDRHB1. CaDRHB1, mainly localized in the nucleus, but also in the cytoplasm in the SUMOylation mimic state, suggesting that SUMOylation of CaDRHB1 promotes its nuclear export, leading to cytoplasmic accumulation. Moreover, CaDSIZ1-silenced pepper plants were less sensitive to ABA and considerably sensitive to drought stress, whereas CaDSIZ1-overexpressing plants displayed ABA-hypersensitive and drought-tolerant phenotypes. Collectively, our data indicate that CaDSIZ1-mediated SUMOylation of CaDRHB1 functions in ABA-mediated drought tolerance.

SUMO E3 ligase SIZ1 connects sumoylation and reactive oxygen species homeostasis processes in Arabidopsis.

The ubiquitin-like modifying peptide SMALL UBIQUITIN-LIKE MODIFIER (SUMO) has become a known modulator of the plant response to multiple environmental stimuli. A common feature of many of these external stresses is the production of reactive oxygen species (ROS). Taking into account that SUMO conjugates rapidly accumulate in response to an external oxidative stimulus, it is likely that ROS and sumoylation converge at the molecular and regulatory levels. In this study, we explored the SUMO-ROS relationship, using as a model the Arabidopsis (Arabidopsis thaliana) null mutant of the major SUMO-conjugation enhancer, the E3 ligase SAP AND MIZ 1 (SIZ1). We showed that SIZ1 is involved in SUMO conjugate increase when primed with both exogenous and endogenous ROS. In siz1, seedlings were sensitive to oxidative stress imposition, and mutants accumulated different ROS throughout development. We demonstrated that the deregulation in hydrogen peroxide and superoxide homeostasis, but not of singlet O2 (1O2), was partially due to SA accumulation in siz1. Furthermore, transcriptomic analysis highlighted a transcriptional signature that implicated siz1 with 1O2 homeostasis. Subsequently, we observed that siz1 displayed chloroplast morphological defects and altered energy dissipation activity and established a link between the chlorophyll precursor protochlorophyllide and deregulation of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA), which is known to drive overproduction of 1O2. Ultimately, network analysis uncovered known and additional associations between transcriptional control of PORA and SIZ1-dependent sumoylation. Our study connects sumoylation, and specifically SIZ1, to the control of chloroplast functions and places sumoylation as a molecular mechanism involved in ROS homeostatic and signaling events.

Dissecting multiple roles of SUMOylation in prostate cancer.

Protein modification with small ubiquitin-like modifiers (SUMOs) plays dual roles in prostate cancer (PCa) tumorigenesis and development. Any intermediary of the SUMO conjugation cycle going awry may forfeit the balance between tumorigenic potential and anticancer effects. Deregulated SUMOylation on the androgen receptor and oncoproteins also takes part in this pathological process, as exemplified by STAT3/NF-κB and tumor suppressors such as PTEN and p53. Here, we outline recent developments and discoveries of SUMOylation in PCa and present an overview of its multiple roles in PCa tumorigenesis/promotion and suppression, while elucidating its potential as a therapeutic target for PCa.

Crosstalk between the chloroplast protein import and SUMO systems revealed through genetic and molecular investigation in Arabidopsis.

The chloroplast proteome contains thousands of different proteins that are encoded by the nuclear genome. These proteins are imported into the chloroplast via the action of the TOC translocase and associated downstream systems. Our recent work has revealed that the stability of the TOC complex is dynamically regulated by the ubiquitin-dependent chloroplast-associated protein degradation pathway. Here, we demonstrate that the TOC complex is also regulated by the small ubiquitin-like modifier (SUMO) system. Arabidopsis mutants representing almost the entire SUMO conjugation pathway can partially suppress the phenotype of ppi1, a pale-yellow mutant lacking the Toc33 protein. This suppression is linked to increased abundance of TOC proteins and improvements in chloroplast development. Moreover, data from molecular and biochemical experiments support a model in which the SUMO system directly regulates TOC protein stability. Thus, we have identified a regulatory link between the SUMO system and the chloroplast protein import machinery.

Novel insights into the impact of the SUMOylation pathway in hematological malignancies (Review)

The small ubiquitin-like modifier (SUMO) system serves an important role in the regulation of protein stability and function. SUMOylation sustains the homeostatic equilibrium of protein function in normal tissues and numerous types of tumor. Accumulating evidence has revealed that SUMO enzymes participate in carcinogenesis via a series of complex cellular or extracellular processes. The present review outlines the physiological characteristics of the SUMOylation pathway and provides examples of SUMOylation participation in different cancer types, including in hematological malignancies (leukemia, lymphoma and myeloma). It has been indicated that the SUMO pathway may influence chromosomal instability, cell cycle progression, apoptosis and chemical drug resistance. The present review also discussed the possible relationship between SUMOylation and carcinogenic mechanisms, and evaluated their potential as biomarkers and therapeutic targets in the diagnosis and treatment of hematological malignancies. Developing and investigating inhibitors of SUMO conjugation in the future may offer promising potential as novel therapeutic strategies.

Viruses, SUMO, and immunity: the interplay between viruses and the host SUMOylation system.

The conjugation of small ubiquitin-like modifier (SUMO) proteins to substrates is a well-described post-translational modification that regulates protein activity, subcellular localization, and protein–protein interactions for a variety of downstream cellular activities. Several studies describe SUMOylation as an essential post-translational modification for successful viral infection across a broad range of viruses, including RNA and DNA viruses, both enveloped and un-enveloped. These viruses include but are not limited to herpes viruses, human immunodeficiency virus-1, and coronaviruses. In addition to the SUMOylation of viral proteins during infection, evidence shows that viruses manipulate the SUMO pathway for host protein SUMOylation. SUMOylation of host and viral proteins greatly impacts host innate immunity through viral manipulation of the host SUMOylation machinery to promote viral replication and pathogenesis. Other post-translational modifications like phosphorylation can also modulate SUMO function. For example, phosphorylation of COUP-TF interacting protein 2 (CTIP2) leads to its SUMOylation and subsequent proteasomal degradation. The SUMOylation of CTIP2 and subsequent degradation prevents CTIP2-mediated recruitment of a multi-enzymatic complex to the HIV-1 promoter that usually prevents the transcription of integrated viral DNA. Thus, the “SUMO switch” could have implications for CTIP2-mediated transcriptional repression of HIV-1 in latency and viral persistence. In this review, we describe the consequences of SUMO in innate immunity and then focus on the various ways that viral pathogens have evolved to hijack the conserved SUMO machinery. Increased understanding of the many roles of SUMOylation in viral infections can lead to novel insight into the regulation of viral pathogenesis with the potential to uncover new targets for antiviral therapies.

Plant SUMO E3 Ligases: Function, Structural Organization, and Connection With DNA.

Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to abiotic stresses. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases accelerate SUMO conjugation while also influencing target identity and interactions. This review explores the biological functions of plant SUMO E3 ligases [SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZs), METHYL METHANESULFONATE-SENSITIVITY PROTEIN 21 (MMS21s), and PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIALs)] in relation to their molecular activities and domains. We also explore the sub-cellular localization of SUMO E3 ligases and review evidence suggesting a connection between certain SUMO E3 ligases and DNA that contributes to gene expression regulation.

SUMO-Modification of Human Nrf2 at K110 and K533 Regulates Its Nucleocytoplasmic Localization, Stability and Transcriptional Activity.

Background/Aims:Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress.Methods:The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell.Results:Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one 532LKDE535 of human (h) and its homologous 524LKDE527 of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K110, in the motifs 109PKSD112 and 109PKQD112 of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m) K110, hK533 and mK525) showed that these residues are needed for SUMO-2 conjugation, nuclear localization and ARE driven transcription of reporter genes and the endogenous HO-1 expression by Nrf2. These residues also stabilized Nrf2, as evident from shorter half-lives of the mutant protein compared to wild-type Nrf2.Conclusion:Our findings indicate that SUMO-2 mediated sumoylation of K110 and K533 in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.

Identification of Protein Direct Interactome with Genetic Code Expansion and Search Engine OpenUaa

Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage. Integrating GECX with OpenUaa, the direct interactome of thioredoxin is identified in Escherichia coli cells, yielding 289 crosslinked peptides and corresponding to 205 direct binding protein of thioredoxin. These identified direct binders provide evidence for thioredoxin's regulation of redox state and mitochondria energy metabolism. When identifying endogenous conjugation of small ubiquitin-like modifier (SUMO), OpenUaa also markedly improves coverage of SUMOylated peptides by ≈92%, revealing new SUMO targets. GECX-OpenUaa will enable efficient identification of direct interactomes of various proteins in live cells.

Histone sumoylation promotes Set3 histone-deacetylase complex-mediated transcriptional regulation.

Histones are substrates of the SUMO (small ubiquitin-like modifier) conjugation pathway. Several reports suggest histone sumoylation affects transcription negatively, but paradoxically, our genome-wide analysis shows the modification concentrated at many active genes. We find that trans-tail regulation of histone-H2B ubiquitylation and H3K4 di-methylation potentiates subsequent histone sumoylation. Consistent with the known control of the Set3 histone deacetylase complex (HDAC) by H3K4 di-methylation, histone sumoylation directly recruits the Set3 complex to both protein-coding and noncoding RNA (ncRNA) genes via a SUMO-interacting motif in the HDAC Cpr1 subunit. The altered gene expression profile caused by reducing histone sumoylation matches well to the profile in cells lacking Set3. Histone H2B sumoylation and the Set3 HDAC coordinately suppress cryptic ncRNA transcription initiation internal to mRNA genes. Our results reveal an elaborate co-transcriptional histone crosstalk pathway involving the consecutive ubiquitylation, methylation, sumoylation and deacetylation of histones, which maintains transcriptional fidelity by suppressing spurious transcription.

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking.

Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.

A Fluorescence-Based Assay to Monitor SUMOylation in Real-Time.

The small ubiquitin-like modifier (SUMO) is an important post-translational modifier that regulates various cellular processes. Extensive investigations have been made to comprehend the enzymatic process and consequence of SUMOylation. In vitro SUMOylation assays are invaluable for understanding the fundamental mechanisms of SUMOylation. A majority of these assays monitor changes in the size of the substrate upon SUMO conjugation. Current methods typically detect the size difference through SDS-PAGE and western blots, which makes these methods cumbersome, error-prone, and time-consuming. Here, we describe a fluorescence-based assay for real-time detection of SUMOylation. In the method, a fluorophore-tagged substrate is used in the SUMOylation reaction. Upon SUMOylation, the size and correlation time (τc ) of the substrate increases, and so does its anisotropy. The rate of change in anisotropy with time reflects the efficiency of the SUMOylation machinery. The real-time SUMOylation assay protocol is elegant, time-saving, and less prone to errors. © 2020 Wiley Periodicals LLC. Basic Protocol: Fluorescent anisotropy-based in vitro SUMOylation assay.