Small Ubiquitin-like Modifier Substrates Research Articles

Rhes, a striatal enriched protein, regulates post-translational small-ubiquitin-like-modifier (SUMO) modification of nuclear proteins and alters gene expression

Rhes (Ras homolog enriched in the striatum), a multifunctional protein that regulates striatal functions associated with motor behaviors and neurological diseases, can shuttle from cell to cell via the formation of tunneling-like nanotubes (TNTs). However, the mechanisms by which Rhes mediates diverse functions remain unclear. Rhes is a small GTPase family member which contains a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes SUMO post-translational modification of proteins (SUMOylation) by promoting “cross-SUMOylation” of the SUMO enzyme SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). Nevertheless, the identity of the SUMO substrates of Rhes remains largely unknown. Here, by combining high throughput interactome and SUMO proteomics, we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene expression. Rhes increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, while decreasing SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 6 different lysine residues (K32, K110, K114, K120, K124, and K245). Furthermore, Rhes regulated the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide evidence for a previously undescribed role for Rhes in regulating the SUMOylation of nuclear targets and in orchestrating striatal gene expression via SUMOylation.

Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors.

Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

The function of SUMOylation and its crucial roles in the development of neurological diseases.

Neurological diseases are relatively complex diseases of a large system; however, the detailed mechanism of their pathogenesis has not been completely elucidated, and effective treatment methods are still lacking for some of the diseases. The SUMO (small ubiquitin-like modifier) modification is a dynamic and reversible process that is catalyzed by SUMO-specific E1, E2, and E3 ligases and reversed by a family of SENPs (SUMO/Sentrin-specific proteases). SUMOylation covalently conjugates numerous cellular proteins, and affects their cellular localization and biological activity in numerous cellular processes. A wide range of neuronal proteins have been identified as SUMO substrates, and the disruption of SUMOylation results in defects in synaptic plasticity, neuronal excitability, and neuronal stress responses. SUMOylation disorders cause many neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. By modulating the ion channel subunit, SUMOylation imbalance is responsible for the development of various channelopathies. The regulation of protein SUMOylation in neurons may provide a new strategy for the development of targeted therapeutic drugs for neurodegenerative diseases and channelopathies.

SUMOylation Connects Cell Stress Responses and Inflammatory Control: Lessons From the Gut as a Model Organ

Conjugation with the small ubiquitin-like modifier (SUMO) constitutes a key post-translational modification regulating the stability, activity, and subcellular localization of its target proteins. However, the vast numbers of identified SUMO substrates obscure a clear view on the function of SUMOylation in health and disease. This article presents a comprehensive review on the physiological relevance of SUMOylation by discussing how global SUMOylation levels—rather than specific protein SUMOylation—shapes the immune response. In particular, we highlight the growing body of work on SUMOylation in intestinal pathologies, because of the unique metabolic, infectious, and inflammatory challenges of this organ. Recent studies show that global SUMOylation can help restrain detrimental inflammation while maintaining immune defenses and tissue integrity. These results warrant further efforts to develop new therapeutic tools and strategies to control SUMOylation in infectious and inflammatory disorders.

2-D08 as a SUMOylation inhibitor induced ROS accumulation mediates apoptosis of acute myeloid leukemia cells possibly through the deSUMOylation of NOX2

Acute myeloid leukemia (AML) is a heterogeneous clonal hematopoietic malignancy with poor survival and frequent relapse. Recently, a posttranslational modification of proteins with small ubiquitin-like modifiers (SUMO) has been notably implicated in a wide spectrum of diseases, especially cancers. Ubc9, as the sole E2-conjugating enzyme in SUMOylation cascade, particularly has been associated with adverse clinical outcomes. 2-D08, a small molecular agent, functions by blocking the transfer of SUMO from the Ubc9 thioester to SUMO substrates without any effects on other individual steps in this process. However, both the effects and mechanisms of 2-D08 on AML cells are still unknown. In this study, we found that 2-D08 significantly suppressed cell viability and colony formation ability. Additionally, it induced mitochondrial-mediated apoptosis with dramatic accumulation of the reactive oxygen species (ROS), which could be almost completely rescued by the ROS scavenger N-acetylcysteine (NAC). Furthermore, we confirmed that the fatal accumulation of ROS was due to its aberrant generation instead of defective scavenging. In summary, our results suggest that 2-D08, as a specific SUMOylation inhibitor, induces ROS accumulation-mediated intrinsic apoptosis of AML cells possibly through deSUMOylation of NOX2. Therefore, 2-D08 might be a promising therapeutic agent for the treatment of AML in the future.

Global analysis of sumoylation function reveals novel insights into development and appressorium-mediated infection of the rice blast fungus.

Protein post-translational modifications play critical roles in cellular processes, development and stress response. The small ubiquitin-like modifier (SUMO) to proteins is one of the essential modifications in eukaryotes, but its function remains largely unknown in plant pathogenic fungi. We present a comprehensive analysis combined with proteomic, molecular and cellular approaches to explore the roles of sumoylation in the model plant fungal pathogen, Magnaporthe oryzae. We found the SUMO pathway plays key roles in colony growth, conidia formation and virulence to the host, as well as cell-cycle-related phenotypes. Sumoylation is also involved in responding to different stresses. Affinity purification identified 940 putative SUMO substrates, many of which were reported to be involved in development, stress response and infection. Interestingly, four septins were also shown to be sumoylated. Mutation of consensus sumoylation sites in each septin all resulted in reduced virulence to the host and dislocation of septins in appressoria. Moreover, sumoylation is also involved in extracellular secretion of different effector proteins. Our study on the functions of sumoylation provides novel insight into development and infection of the rice blast fungus.

Identification of SUMO conjugation sites in the budding yeast proteome.

Post-translational modification by the small ubiquitin-like modifier (SUMO) is an important mechanism regulating protein function. Identification of SUMO conjugation sites on substrates is a challenging task. Here we employed a proteomic method to map SUMO acceptor lysines in budding yeast proteins. We report the identification of 257 lysine residues where SUMO is potentially attached. Amongst the hits, we identified already known SUMO substrates and sites, confirming the success of the approach. In addition, we tested several of the novel substrates using SUMO immunoprecipitation analysis and confirmed that the SUMO acceptor lysines identified in these proteins are indeed bona fide SUMOylation sites. We believe that the collection of SUMO sites presented here is an important resource for future functional studies of SUMOylation in yeast.

Protein SUMOylation is Involved in Cell-cycle Progression and Cell Morphology in Giardia lamblia.

The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.

Targeting of SUMO substrates to a Cdc48-Ufd1-Npl4 segregase and STUbL pathway in fission yeast.

In eukaryotes, the conjugation of proteins to the small ubiquitin-like modifier (SUMO) regulates numerous cellular functions. A proportion of SUMO conjugates are targeted for degradation by SUMO-targeted ubiquitin ligases (STUbLs) and it has been proposed that the ubiquitin-selective chaperone Cdc48/p97-Ufd1-Npl4 facilitates this process. However, the extent to which the two pathways overlap, and how substrates are selected, remains unknown. Here we address these questions in fission yeast through proteome-wide analyses of SUMO modification sites. We identify over a thousand sumoylated lysines in a total of 468 proteins and quantify changes occurring in the SUMO modification status when the STUbL or Ufd1 pathways are compromised by mutations. The data suggest the coordinated processing of several classes of SUMO conjugates, many dynamically associated with centromeres or telomeres. They provide new insights into subnuclear organization and chromosome biology, and, altogether, constitute an extensive resource for the molecular characterization of SUMO function and dynamics.

Global Reprogramming of Host SUMOylation during Influenza Virus Infection.

SummaryDynamic nuclear SUMO modifications play essential roles in orchestrating cellular responses to proteotoxic stress, DNA damage, and DNA virus infection. Here, we describe a non-canonical host SUMOylation response to the nuclear-replicating RNA pathogen, influenza virus, and identify viral RNA polymerase activity as a major contributor to SUMO proteome remodeling. Using quantitative proteomics to compare stress-induced SUMOylation responses, we reveal that influenza virus infection triggers unique re-targeting of SUMO to 63 host proteins involved in transcription, mRNA processing, RNA quality control, and DNA damage repair. This is paralleled by widespread host deSUMOylation. Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes. Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression. Our global characterization of influenza virus-triggered SUMO redistribution provides a proteomic resource to understand host nuclear SUMOylation responses to infection.

SUMOylation of Argonaute-2 regulates RNA interference activity

Post-translational modification of substrate proteins by small ubiquitin-like modifier (SUMO) regulates a vast array of cellular processes. SUMOylation occurs through three sequential enzymatic steps termed E1, E2 and E3. Substrate selection can be determined through interactions between the target protein and the SUMO E2 conjugating enzyme Ubc9 and specificity can be enhanced by substrate interactions with E3 ligase enzymes. We used the putative substrate recognition (PINIT) domain from the SUMO E3 PIAS3 as bait to identify potential SUMO substrates. One protein identified was Argonaute-2 (Ago2), which mediates RNA-induced gene silencing through binding small RNAs and promoting degradation of complimentary target mRNAs. We show that Ago2 can be SUMOylated in mammalian cells by both SUMO1 and SUMO2. SUMOylation occurs primarily at K402, and mutation of the SUMO consensus site surrounding this lysine reduces Ago2-mediated siRNA-induced silencing in a luciferase-based reporter assay. These results identify SUMOylation as a potential regulator of Ago2 activity and open new avenues for research into the mechanisms underlying the regulation of RNA-induced gene silencing.

SUMOylation Regulates Polo-like Kinase 1-interacting Checkpoint Helicase (PICH) during Mitosis

Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.

GPS-SUMO: a tool for the prediction of sumoylation sites and SUMO-interaction motifs.

Small ubiquitin-like modifiers (SUMOs) regulate a variety of cellular processes through two distinct mechanisms, including covalent sumoylation and non-covalent SUMO interaction. The complexity of SUMO regulations has greatly hampered the large-scale identification of SUMO substrates or interaction partners on a proteome-wide level. In this work, we developed a new tool called GPS-SUMO for the prediction of both sumoylation sites and SUMO-interaction motifs (SIMs) in proteins. To obtain an accurate performance, a new generation group-based prediction system (GPS) algorithm integrated with Particle Swarm Optimization approach was applied. By critical evaluation and comparison, GPS-SUMO was demonstrated to be substantially superior against other existing tools and methods. With the help of GPS-SUMO, it is now possible to further investigate the relationship between sumoylation and SUMO interaction processes. A web service of GPS-SUMO was implemented in PHP + JavaScript and freely available at http://sumosp.biocuckoo.org.

A SUMO Atlas shoulders the DNA damage response (477.3)

The maintenance of genomic integrity requires multiple types of protein modification that are readily reversible and therefore capable of dynamic signaling and regulation. Our lab has been studying how protein modification by SUMO (small ubiquitin‐like modifier) contributes to the maintenance of the genome. Studies from our lab and others have shown that mutating sumoylation and desumoylation enzymes results in multiple defects in DNA repair and chromosomal segregation. For example, mutating the sumoylation E2 and the Mms21 E3 enzymes in yeast leads to the accumulation of toxic recombination intermediates when cells replicate over damaged templates. Some of these defects can be partly explained by the lack of modification of known DNA metabolism substrates, such as PCNA, Rad52, and TDG, sumoylation of which can modulate their DNA repair functions. However, majority of the phenotypes seen in mutants of the sumoylation enzymes cannot be explained by the lack of sumoylation of the previously known SUMO substrates. This indicates the existence of additional targets that contribute to the roles of sumoylation in genome maintenance. We have undertaken a biochemical approach to identify new sumoylation substrates and have examined how sumoylation contributes to the cellular response to DNA damage, both at a global level and at the individual substrate level. Our results indicate the existence of an extensive DNA damage‐induced sumoylation system that makes multiple contributions to genome stability and operates largely independent of the canonical DNA damage checkpoint kinase. Our progress to understand this new system and its effects will be reported and discussed.

Tomosyn Interacts with the SUMO E3 Ligase PIASγ

Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASγ (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASγ. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASγ-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment.

In vivo characterization of the properties of SUMO1-specific monobodies

Monobodies are small recombinant proteins designed to bind with high affinity to target proteins. Monobodies have been generated to mimic the SIM [SUMO (small ubiquitin-like modifier)-interacting motif] present in many SUMO target proteins, but their properties have not been determined in cells. In the present study we characterize the properties of two SUMO1-specific monobodies (hS1MB4 and hS1MB5) in HEK (human embyronic kidney)-293 and HeLa cells and examine their ability to purify SUMO substrates from cell lines and rat brain. Both hS1MB4 and hS1MB5 compared favourably with commercially available antibodies and were highly selective for binding to SUMO1 over SUMO2/3 in pull-down assays against endogenous and overexpressed SUMO and SUMOylated proteins. Monobodies expressed in HeLa cells displayed a nuclear and cytosolic distribution that overlaps with SUMO1. Expression of the monobodies effectively inhibited protein SUMOylation by SUMO1 and, surprisingly, by SUMO2/3, but were not cytotoxic for at least 36 h. We attribute the effects on SUMO2/3 to the role of SUMO1 in chain termination and/or monobody inhibition of the SUMO-conjugating E1 enzyme complex. Taken together, these data provide the first demonstration that monobodies represent useful new tools both to isolate SUMO conjugates and to probe cell SUMOylation pathways in vivo.

SUMO and Alzheimer’s Disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and is the most common cause of dementia in the elderly. Histopathologically, AD features insoluble aggregates of two proteins in the brain, amyloid-β (Aβ) and the microtubule-associated protein tau, both of which have been linked to the small ubiquitin-like modifier (SUMO). A large body of research has elucidated many of the molecular and cellular pathways that underlie AD, including those involving the abnormal Aβ and tau aggregates. However, a full understanding of the etiology and pathogenesis of the disease has remained elusive. Consequently, there are currently no effective therapeutic options that can modify the disease progression and slow or stop the decline of cognitive functioning. As part of the effort to address this lacking, there needs a better understanding of the signaling pathways that become impaired under AD pathology, including the regulatory mechanisms that normally control those networks. One such mechanism involves SUMOylation, which is a post-translational modification (PTM) that is involved in regulating many aspects of cell biology and has also been found to have several critical neuron-specific roles. Early studies have indicated that the SUMO system is likely altered with AD-type pathology, which may impact Aβ levels and tau aggregation. Although still a relatively unexplored topic, SUMOylation will likely emerge as a significant factor in AD pathogenesis in ways which may be somewhat analogous to other regulatory PTMs such as phosphorylation. Thus, in addition to the upstream effects on tau and Aβ processing, there may also be downstream effects mediated by Aβ aggregates or other AD-related factors on SUMO-regulated signaling pathways. Multiple proteins that have functions relevant to AD pathology have been identified as SUMO substrates, including those involved in synaptic physiology, mitochondrial dynamics, and inflammatory signaling. Ongoing studies will determine how these SUMO-regulated functions in neurons and glial cells may be impacted by Aβ and AD pathology. Here, we present a review of the current literature on the involvement of SUMO in AD, as well as an overview of the SUMOylated proteins and pathways that are potentially dysregulated with AD pathogenesis.

Quantitative Proteomics Reveals Factors Regulating RNA Biology as Dynamic Targets of Stress-induced SUMOylation in Arabidopsis

The stress-induced attachment of small ubiquitin-like modifier (SUMO) to a diverse collection of nuclear proteins regulating chromatin architecture, transcription, and RNA biology has been implicated in protecting plants and animals against numerous environmental challenges. In order to better understand stress-induced SUMOylation, we combined stringent purification of SUMO conjugates with isobaric tag for relative and absolute quantification mass spectrometry and an advanced method to adjust for sample-to-sample variation so as to study quantitatively the SUMOylation dynamics of intact Arabidopsis seedlings subjected to stress. Inspection of 172 SUMO substrates during and after heat shock (37 °C) revealed that stress mostly increases the abundance of existing conjugates, as opposed to modifying new targets. Some of the most robustly up-regulated targets participate in RNA processing and turnover and RNA-directed DNA modification, thus implicating SUMO as a regulator of the transcriptome during stress. Many of these targets were also strongly SUMOylated during ethanol and oxidative stress, suggesting that their modification is crucial for general stress tolerance. Collectively, our quantitative data emphasize the importance of SUMO to RNA-related processes protecting plants from adverse environments.

SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.

Identification of protein SUMOylation sites by mass spectrometry using combined microwave‐assisted aspartic acid cleavage and tryptic digestion

Small-ubiquitin-like modifier (SUMO) is a posttranslational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization and mitosis, among other cellular processes, are known to occur. However, although the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly "tag" that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32-amino-acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches, which serve to shorten the SUMO tag and thus help to simplify SUMO spectra, making interpretation of mass spectra and location of the SUMOylation site easier. As described in this report, we demonstrate a method for identifying SUMOylation sites using three commercially available SUMO- modified isoforms and by employing acid-only and acid/trypsin cleavage strategies. These approaches were carried out using MALDI-time-of-flight (TOF) and LC/MS instrumentation, along with collision induced dissociation (CID) and electron transfer dissociation (ETD).