Molecular Characterization and Functional Localization of a Novel SUMOylation Gene in Oryza sativa

Small ubiquitin-related modifier (SUMO) regulates the cellular function of diverse proteins through post-translational modifications. The current study defined a new homolog of SUMO genes in the rice genome and named it OsSUMO7. Putative protein analysis of OsSUMO7 detected SUMOylation features, including di-glycine (GG) and consensus motifs (ΨKXE/D) for the SUMOylation site. Phylogenetic analysis demonstrated the high homology of OsSUMO7 with identified rice SUMO genes, which indicates that the OsSUMO7 gene is an evolutionarily conserved SUMO member. RT-PCR analysis revealed that OsSUMO7 was constitutively expressed in all plant organs. Bioinformatic analysis defined the physicochemical properties and structural model prediction of OsSUMO7 proteins. A red fluorescent protein (DsRed), fused with the OsSUMO7 protein, was expressed and localized mainly in the nucleus and formed nuclear subdomain structures. The fusion proteins of SUMO-conjugating enzymes with the OsSUMO7 protein were co-expressed and co-localized in the nucleus and formed nuclear subdomains. This indicated that the OsSUMO7 precursor is processed, activated, and transported to the nucleus through the SUMOylation system of the plant cell.

CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding

CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε32 and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε27) as an antagonist of GATA-1 transactivation of the eosinophil’s major basic protein-1 (MBP1) P2-promoter, showing C/EBPε27 and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε27 fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε27 is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε27 mapping its repressor domains, comparing it to C/EBPε32 and C/EBPε14, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε27 repression of GATA-1 is mediated by its unique 68aa N-terminus combined with previously identified RDI domain. This repressor activity does not require, but is enhanced by, DNA binding via the basic region of C/EBPε27 but independent of sumoylation of the RDI core “VKEEP” sumoylation site. These findings identify the N-terminus of C/EBPε27 as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε27 repression of GATA-1 occurs via a combination of both C/EBPε27-GATA-1 protein–protein interaction and C/EBPε27 binding to a C/EBP site in the MBP1 promoter. The C/EBPε27 isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation, as these genes are ultimately silenced in the mature cell. Understanding the functionality of C/EBPε27 in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.

An Ensemble Deep Learning based Predictor for Simultaneously Identifying Protein Ubiquitylation and SUMOylation Sites

Background Several computational tools for predicting protein Ubiquitylation and SUMOylation sites have been proposed to study their regulatory roles in gene location, gene expression, and genome replication. However, existing methods generally rely on feature engineering, and ignore the natural similarity between the two types of protein translational modification. This study is the first all-in-one deep network to predict protein Ubiquitylation and SUMOylation sites from protein sequences as well as their crosstalk sites simultaneously. Our deep learning architecture integrates several meta classifiers that apply deep neural networks to protein sequence information and physico-chemical properties, which were trained on multi-label classification mode for simultaneously identifying protein Ubiquitylation and SUMOylation as well as their crosstalk sites. Results The promising AUCs of our method on Ubiquitylation, SUMOylation and crosstalk sites achieved 0.838, 0.888, and 0.862 respectively on tenfold cross-validation. The corresponding APs reached 0.683, 0.804 and 0.552, which also validated our effectiveness. Conclusions The proposed architecture managed to classify ubiquitylated and SUMOylated lysine residues along with their crosstalk sites, and outperformed other well-known Ubiquitylation and SUMOylation site prediction tools.

Recent development of machine learning methods in sumoylation sites prediction

: Sumoylation of proteins is an important reversible post-translational modification of proteins and mediates a variety of cellular processes. Sumo-modified proteins can change their subcellular localization, activity and stability. In addition, it also plays an important role in various cellular processes such as transcriptional regulation and signal transduction. The abnormal sumoylation is involved in many diseases, including neurodegeneration and immune-related diseases, as well as the development of cancer. Therefore, identification of the sumoylation site (SUMO site) is fundamental to understanding their molecular mechanisms and regulatory roles. In contrast to labor-intensive and costly experimental approaches, computational prediction of sumoylation sites in silico also attracted much attention for its accuracy, convenience and speed. At present, many computational prediction models have been used to identify SUMO sites, but these contents have not been comprehensively summarized and reviewed. Therefore, the research progress of relevant models is summarized and discussed in this paper. We will briefly summarize the development of bioinformatics methods on sumoylation site prediction. We will mainly focus on the benchmark dataset construction, feature extraction, machine learning method, published results and online tools. We hope the review will provide more help for wet-experimental scholars.

Drosophila H2Av negatively regulates the activity of the IMD pathway via facilitating Relish SUMOylation

Insects depend on the innate immune response for defense against a wide array of pathogens. Central to Drosophila immunity are antimicrobial peptides (AMPs), released into circulation when pathogens trigger either of the two widely studied signal pathways, Toll or IMD. The Toll pathway responds to infection by Gram-positive bacteria and fungi while the IMD pathway is activated by Gram-negative bacteria. During activation of the IMD pathway, the NF-κB-like transcription factor Relish is phosphorylated and then cleaved, which is crucial for IMD-dependent AMP gene induction. Here we show that loss-of-function mutants of the unconventional histone variant H2Av upregulate IMD-dependent AMP gene induction in germ-free Drosophila larvae and adults. After careful dissection of the IMD pathway, we found that Relish has an epistatic relationship with H2Av. In the H2Av mutant larvae, SUMOylation is down-regulated, suggesting a possible role of SUMOylation in the immune phenotype. Eventually we demonstrated that Relish is mostly SUMOylated on amino acid K823. Loss of the potential SUMOylation site leads to significant auto-activation of Relish in vivo. Further work indicated that H2Av regulates Relish SUMOylation after physically interacting with Su(var)2-10, the E3 component of the SUMOylation pathway. Biochemical analysis suggested that SUMOylation of Relish prevents its cleavage and activation. Our findings suggest a new mechanism by which H2Av can negatively regulate, and thus prevent spontaneous activation of IMD-dependent AMP production, through facilitating SUMOylation of the NF-κB like transcription factor Relish.

iSUMOK-PseAAC: prediction of lysine sumoylation sites using statistical moments and Chou’s PseAAC

Sumoylation is the post-translational modification that is involved in the adaption of the cells and the functional properties of a large number of proteins. Sumoylation has key importance in subcellular concentration, transcriptional synchronization, chromatin remodeling, response to stress, and regulation of mitosis. Sumoylation is associated with developmental defects in many human diseases such as cancer, Huntington’s, Alzheimer’s, Parkinson’s, Spin cerebellar ataxia 1, and amyotrophic lateral sclerosis. The covalent bonding of Sumoylation is essential to inheriting part of the operative characteristics of some other proteins. For that reason, the prediction of the Sumoylation site has significance in the scientific community. A novel and efficient technique is proposed to predict the Sumoylation sites in proteins by incorporating Chou’s Pseudo Amino Acid Composition (PseAAC) with statistical moments-based features. The outcomes from the proposed system using 10 fold cross-validation testing are 94.51%, 94.24%, 94.79% and 0.8903% accuracy, sensitivity, specificity and MCC, respectively. The performance of the proposed system is so far the best in comparison to the other state-of-the-art methods. The codes for the current study are available on the GitHub repository using the link: https://github.com/csbioinfopk/iSumoK-PseAAC.

Sumoylation of the Carboxy-Terminal of Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Attenuates Viral DNA Replication

Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.

Viral RNA-binding ability conferred by SUMOylation at PB1 K612 of influenza A virus is essential for viral pathogenesis and transmission

Posttranslational modifications, such as SUMOylation, play specific roles in the life cycle of invading pathogens. However, the effect of SUMOylation on the adaptation, pathogenesis, and transmission of influenza A virus (IAV) remains largely unknown. Here, we found that a conserved lysine residue at position 612 (K612) of the polymerase basic protein 1 (PB1) of IAV is a bona fide SUMOylation site. SUMOylation of PB1 at K612 had no effect on the stability or cellular localization of PB1, but was critical for viral ribonucleoprotein (vRNP) complex activity and virus replication in vitro. When tested in vivo, we found that the virulence of SUMOylation-defective PB1/K612R mutant IAVs was highly attenuated in mice. Moreover, the airborne transmission of a 2009 pandemic H1N1 PB1/K612R mutant virus was impaired in ferrets, resulting in reversion to wild-type PB1 K612. Mechanistically, SUMOylation at K612 was essential for PB1 to act as the enzymatic core of the viral polymerase by preserving its ability to bind viral RNA. Our study reveals an essential role for PB1 K612 SUMOylation in the pathogenesis and transmission of IAVs, which can be targeted for the design of anti-influenza therapies.

SUMOylome Modulates Intraflagellar Transport Machinery and Eukaryotic Cilia Motility

Translocation of channelrhodopsins (ChRs) is mediated by intraflagellar transport (IFT) machinery. However, the functional role of the network containing photoreceptors, IFT and other proteins in controlling cilia motility of the alga is still not fully delineated. In the current study, we identified two important motifs at the C-terminus of ChR1. One of them is similar to a known ciliary targeting sequence that specifically interacts with a small GTPase, and the other is a SUMOylation site. For the first time, experimental data provide an insight into the role of SUMOylation in the modulation of IFT & ChR1. Blocking of SUMOylation affected the phototaxis of C. reinhardtii cells. This implies SUMOylation based regulation of protein network controlling photomotility. The conservation of SUMOylation site pattern as analyzed for the relevant photoreceptors, IFT and its associated signaling proteins in other ciliated green algae suggested SUMOylation based photobehavioural response across the microbes. This report establishes a link between evolutionary conserved SUMOylation and ciliary machinery for the maintenance and functioning of cilia across the eukaryotes. Our enriched SUMOylome of C. reinhardtii comprehends the proteins related to ciliary development and, photo-signaling, along with homologue(s) associated to human ciliopathies as SUMO targets.

SUMOylation contributes to proteostasis of the chloroplast protein import receptor TOC159 during early development

Chloroplast biogenesis describes the transition of non-photosynthetic proplastids to photosynthetically active chloroplasts in the cells of germinating seeds. Chloroplast biogenesis requires the import of thousands of nuclear-encoded preproteins by essential import receptor TOC159. We demonstrate that the small ubiquitin-related modifier (SUMO) pathway crosstalks with the ubiquitin–proteasome pathway to affect TOC159 stability during early plant development. We identified a SUMO3-interacting motif (SIM) in the TOC159 GTPase domain and a SUMO3 covalent SUMOylation site in the membrane domain. A single K to R substitution (K1370R) in the M-domain disables SUMOylation. Compared to wild-type TOC159, TOC159K1370R was destabilized under UPS-inducing stress conditions. However, TOC159K1370R recovered to same protein level as wild-type TOC159 in the presence of a proteasome inhibitor. Thus, SUMOylation partially stabilizes TOC159 against UPS-dependent degradation under stress conditions. Our data contribute to the evolving model of tightly controlled proteostasis of the TOC159 import receptor during proplastid to chloroplast transition.