Xiang Dong Zhang

SUMO-2/3 Modification and Binding Regulate the Association of CENP-E with Kinetochores and Progression through Mitosis

SUMOylation is essential for cell-cycle regulation in invertebrates; however, its functions during the mammalian cell cycle are largely uncharacterized. Mammals express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are 96% identical and referred to as SUMO-2/3). We found that SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to the mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. Consistent with this, global inhibition of SUMOylation caused a prometaphase arrest due to defects in targeting the microtubule motor protein CENP-E to kinetochores. CENP-E was found to be modified specifically by SUMO-2/3 and to possess SUMO-2/3 polymeric chain-binding activity essential for kinetochore localization. Our findings indicate that SUMOylation is a key regulator of the mammalian cell cycle, with SUMO-1 and SUMO-2/3 modification of different proteins regulating distinct processes.

Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software

Tandem mass spectrometry (MS/MS) allows for the rapid identification of many types of post-translational modifications (PTMs), especially those that can be detected by a diagnostic mass shift in one or more peptide fragment ions (for example, phosphorylation). But some PTMs (for example, SUMOs and other ubiquitin-like modifiers) themselves produce multiple fragment ions; combined with fragments from the modified target peptide, a complex overlapping fragmentation pattern is thus generated, which is uninterpretable by standard peptide sequencing software. Here we introduce SUMmOn, an automated pattern recognition tool that detects diagnostic PTM fragment ion series within complex MS/MS spectra, to identify modified peptides and modification sites within these peptides. Using SUMmOn, we demonstrate for the first time that human SUMO-1 multimerizes in vitro primarily via three N-terminal lysines, Lys7, Lys16 and Lys17. Notably, our method is theoretically applicable to any type of modification or chemical moiety generating a unique fragment ion pattern.

Developmental control of sumoylation pathway proteins in mouse male germ cells

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.

Protection from isopeptidase-mediated deconjugation regulates paralog-selective sumoylation of RanGAP1.

Vertebrates express three small ubiquitin-related modifiers (SUMO-1, SUMO-2, and SUMO-3) that are conjugated in part to unique subsets of proteins and, thereby, regulate distinct cellular processes. Mechanisms regulating paralog-selective sumoylation, however, remain poorly understood. Despite being equally well modified by SUMO-1 and SUMO-2 in vitro, RanGAP1 is selectively modified by SUMO-1 in vivo. We have found that this paralog-selective modification is determined at the level of deconjugation by isopeptidases. Our findings indicate that, relative to SUMO-2-modified RanGAP1, SUMO-1-modified RanGAP1 forms a more stable, higher affinity complex with the nucleoporin Nup358/RanBP2 that preferentially protects it from isopeptidases. By swapping residues in SUMO-1 and SUMO-2 responsible for Nup358/RanBP2 binding, or by manipulating isopeptidase expression levels, paralog-selective modification of RanGAP1 could be affected both in vitro and in vivo. Thus, protection from isopeptidases, through interactions with SUMO-binding proteins, represents an important mechanism defining paralog-selective sumoylation.

SUMOylation in Control of Accurate Chromosome Segregation during Mitosis

Posttranslational protein modification by small ubiquitin-related modifier (SUMO) has emerged as an important regulatory mechanism for chromosome segregation during mitosis. This review focuses on how SUMOylation regulates the centromere and kinetochore activities to achieve accurate chromosome segregation during mitosis. Kinetochores are assembled on the specialized chromatin domains called centromeres and serve as the sites for attaching spindle microtubule to segregate sister chromatids to daughter cells. Many proteins associated with mitotic centromeres and kinetochores have been recently found to be modified by SUMO. Although we are still at the early stage of elucidating how SUMOylation controls chromosome segregation during mitosis, a substantial progress has been achieved over the past decade. Furthermore, a major theme that has emerged from the recent studies of SUMOylation in mitosis is that both SUMO conjugation and deconjugation are critical for kinetochore assembly and disassembly. Lastly, we propose a model that SUMOylation coordinates multiple centromere and kinetochore activities to ensure accurate chromosome segregation.

An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages

Ubiquitin (Ub) and the ubiquitin‐like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half‐life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis‐database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine‐rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin‐related modifier (SUMO) chain (SUMO‐2 K42, SUMO‐3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.

Identification of SUMO‐2/3‐modified proteins associated with mitotic chromosomes

Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO‐2/3 (small ubiquitin‐related modifier) to the inner centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO‐2/3 from mitotic chromosomes. Using these methods, we identified 149 chromosome‐associated SUMO‐2/3 substrates by nLC‐ESI‐MS/MS. Approximately one‐third of the identified proteins have reported functions in mitosis. Consistent with SUMO‐2/3 immunolocalization, we identified known centromere‐ and kinetochore‐associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs.

Analysis of changes in SUMO-2/3 modification during breast cancer progression and metastasis.

SUMOylation is an essential posttranslational modification and regulates many cellular processes. Dysregulation of SUMOylation plays a critical role in metastasis, yet how its perturbation affects this lethal process of cancer is not well understood. We found that SUMO-2/3 modification is greatly up-regulated in metastatic breast cancer cells compared with nonmetastatic control cells. To identify proteins differentially modified by SUMO-2/3 between metastatic and nonmetastatic cells, we established a method in which endogenous SUMO-2/3 conjugates are labeled by stable isotope labeling by amino acids in cell culture (SILAC), immunopurified by SUMO-2/3 monoclonal antibodies and epitope-peptide elution, and analyzed by quantitative mass spectrometry. We identified 66 putative SUMO-2/3-conjugated proteins, of which 15 proteins show a significant increase/decrease in SUMO-2/3 modification in metastatic cells. Targets with altered SUMOylation are involved in cell cycle, migration, inflammation, glycolysis, gene expression, and SUMO/ubiquitin pathways, suggesting that perturbations of SUMO-2/3 modification might contribute to metastasis by affecting these processes. Consistent with this, up-regulation of PML SUMO-2/3 modification corresponds to an increased number of PML nuclear bodies (PML-NBs) in metastatic cells, whereas up-regulation of global SUMO-2/3 modification promotes 3D cell migration. Our findings provide a foundation for further investigating the effects of SUMOylation on breast cancer progression and metastasis.

Ub in charge: Regulating E2 enzyme nuclear import

The activation and targeted localization of ubiquitin E2 conjugating enzymes could provide a point for regulating ubiquitin-dependent cell functions. Supporting this view, the ubiquitin charging and activation of a class of E2 enzymes has been directly linked to their nuclear import.

Molecular characterization and functional analysis of annulate lamellae pore complexes in nuclear transport in mammalian cells

Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the interaction between annulate lamellae pore complexes and nuclear transport complexes in mammalian cells.