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Identification of small ubiquitin-like modifier substrates with diverse functions using the Xenopus egg extract system.
Ma L, Aslanian A, Sun H, Jin M, Shi Y, Yates JR, Hunter T.
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Post-translational modification by SUMO is a highly conserved pathway in eukaryotes that plays very important regulatory roles in many cellular processes. Deregulation of the SUMO pathway contributes to the development and progression of many diseases including cancer. Therefore, identifying additional SUMO substrates and studying how their cellular and biological functions are regulated by sumoylation should provide new insights. Our studies showed that sumoylation activity was significant in Xenopus egg extracts, and that a high level of sumoylation was associated with sperm chromatin when SUMO was incubated with Xenopus egg extracts. By isolating SUMO-conjugated substrates using His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified 346 proteins by mass spectrometry analysis that were not present in control pull-downs. Among them, 167 proteins were identified from interphase egg extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and 228 proteins by both. We validated the sumoylation of five candidates, CKB, ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in this study were involved in diverse biological processes. Additionally, SUMO substrates identified from different cell cycle stages or pulled down by different SUMO homologs were enriched for distinct cellular components and functional categories. Our results comprehensively profile the sumoylation occurring in the Xenopus egg extract system.
Fig. 2. C
C, SUMO1 could be conjugated to sperm chromatin. Sperm chromatin isolated from male Xenopus was incubated with interphase Xenopus egg extracts and purified His-GFP-tagged SUMO1 mutant proteins. A strong GFP signal appeared on the chromatin in Xenopus egg extracts in less than 10 min and persisted even after the nuclear formation in WT and HFV groups. No GFP signal was observed on sperm chromatin with the AA or HFV-AA SUMO mutants. Microtubule structures formed by rhodamine-labeled tubulin indicate the cell cycle stage of Xenopus egg extracts. DAPI was used for DNA staining. Scale bar equals 10um
Fig. 2D, SUMO2 could be conjugated on sperm chromatin. Sperm chromatin isolated from male Xenopus was incubated with interphase Xenopus egg extracts and purified His-GFP-tagged SUMO2 mutant proteins. A strong GFP signal appeared on the chromatin with WT and QFI SUMO. No GFP signal was observed with the AA or QFI-AA SUMO mutants. Microtubule structures formed by rhodamine-labeled tubulin indicate the cell cycle stage of Xenopus egg extracts. DAPI was used for DNA staining. Scale bar equals 10um
FIG. 5. Functional analysis of SUMO substrates. A, Pie chart showing gene ontology analysis of the SUMO candidate proteins. The genes were grouped into categories defined by GO Biological Processes by the PANTHER classification system. The largest category is that containing gene products that play roles in metabolic process.
FIG. 5. Functional analysis of SUMO substrates.
B, Bar chart showing enrichment p values of SUMO substrates identified from interphase or mitotic phase for GO Biological Process terms generated by DAVID database. C, Bar chart showing enrichment p values of SUMO1 and SUMO2 substrates for GO Biological Process terms generated by DAVID database.
FIG. 6. Functional protein interaction network analysis based on the STRING database. The image illustrates a network analysis of sumoylated proteins that was created with STRING. Proteins isolated from mitotic phase, interphase or both were highlighted in red, green or white, respectively. The protein complexes and pathways highlighted by red circle are involved in: (1) ribosome assembly and translation; (2) proteasomal degradation; (3) DNA replication and repair; (4) chromatin remodeling and transcriptional regulation; (5) cell cycle regulation; (6) the mitochondrial respiratory chain; (7) metabolic regulation; and (8) the sumoylation pathway.
Alarcon-Vargas,
SUMO in cancer--wrestlers wanted.
2002, Pubmed
Alarcon-Vargas,
SUMO in cancer--wrestlers wanted.
2002,
Pubmed Ayaydin,
Distinct in vivo dynamics of vertebrate SUMO paralogues.
2004,
Pubmed Azuma,
SUMO-2/3 regulates topoisomerase II in mitosis.
2003,
Pubmed
,
Xenbase Bessman,
The creatine-creatine phosphate energy shuttle.
1985,
Pubmed Bonne-Andrea,
SUMO2/3 modification of cyclin E contributes to the control of replication origin firing.
2013,
Pubmed
,
Xenbase Bruderer,
Purification and identification of endogenous polySUMO conjugates.
2011,
Pubmed Creton,
SnapShot: The SUMO system.
2010,
Pubmed Dawlaty,
Resolution of sister centromeres requires RanBP2-mediated SUMOylation of topoisomerase IIalpha.
2008,
Pubmed Eng,
An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.
1994,
Pubmed Flotho,
Sumoylation: a regulatory protein modification in health and disease.
2013,
Pubmed Franceschini,
STRING v9.1: protein-protein interaction networks, with increased coverage and integration.
2013,
Pubmed Galisson,
A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells.
2011,
Pubmed Geiss-Friedlander,
Concepts in sumoylation: a decade on.
2007,
Pubmed Guo,
Proteomic analysis of SUMO4 substrates in HEK293 cells under serum starvation-induced stress.
2005,
Pubmed Hannak,
Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts.
2006,
Pubmed
,
Xenbase Hayashi,
Ubc9 is essential for viability of higher eukaryotic cells.
2002,
Pubmed Huang,
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
2009,
Pubmed Ishihama,
Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.
2005,
Pubmed Johnson,
The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.
1997,
Pubmed Joseph,
SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles.
2002,
Pubmed Kessler,
A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis.
2012,
Pubmed Kim,
Systematic and quantitative assessment of the ubiquitin-modified proteome.
2011,
Pubmed Knuesel,
A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program.
2005,
Pubmed Leach,
Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts.
2005,
Pubmed
,
Xenbase Lohka,
Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.
1985,
Pubmed
,
Xenbase Ma,
Requirement for Nudel and dynein for assembly of the lamin B spindle matrix.
2009,
Pubmed
,
Xenbase Mahajan,
Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association.
1998,
Pubmed März,
Ataxin-10, the spinocerebellar ataxia type 10 neurodegenerative disorder protein, is essential for survival of cerebellar neurons.
2004,
Pubmed Matic,
Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif.
2010,
Pubmed McDonald,
MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications.
2004,
Pubmed Medema,
Boosting and suppressing mitotic phosphorylation.
2011,
Pubmed Melchior,
SUMO-1 and p53.
2002,
Pubmed Mi,
PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees.
2013,
Pubmed Mudunuri,
bioDBnet: the biological database network.
2009,
Pubmed Panse,
A proteome-wide approach identifies sumoylated substrate proteins in yeast.
2004,
Pubmed Peng,
Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.
2003,
Pubmed Powers,
Preparation and use of interphase Xenopus egg extracts.
2001,
Pubmed
,
Xenbase Psakhye,
Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair.
2012,
Pubmed Ryu,
PIASy mediates SUMO-2/3 conjugation of poly(ADP-ribose) polymerase 1 (PARP1) on mitotic chromosomes.
2010,
Pubmed
,
Xenbase Saitoh,
Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3.
2000,
Pubmed Saitoh,
Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.
1998,
Pubmed
,
Xenbase Schimmel,
Uncovering SUMOylation dynamics during cell-cycle progression reveals FoxM1 as a key mitotic SUMO target protein.
2014,
Pubmed Shiio,
Histone sumoylation is associated with transcriptional repression.
2003,
Pubmed Sumara,
E3 ubiquitin ligases and mitosis: embracing the complexity.
2008,
Pubmed Sun,
Poly-small ubiquitin-like modifier (PolySUMO)-binding proteins identified through a string search.
2012,
Pubmed Sun,
Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins.
2007,
Pubmed Tabb,
DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics.
2002,
Pubmed Tsai,
A mitotic lamin B matrix induced by RanGTP required for spindle assembly.
2006,
Pubmed
,
Xenbase Ubersax,
Targets of the cyclin-dependent kinase Cdk1.
2003,
Pubmed Vertegaal,
Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics.
2006,
Pubmed Vethantham,
In vitro sumoylation of recombinant proteins and subsequent purification for use in enzymatic assays.
2009,
Pubmed Vigodner,
Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa.
2013,
Pubmed Watts,
The role of SUMO in chromosome segregation.
2007,
Pubmed Xu,
Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.
2010,
Pubmed Zhang,
SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis.
2008,
Pubmed
,
Xenbase Zhao,
Sumoylation regulates diverse biological processes.
2007,
Pubmed Zheng,
Sequencing and expression of complementary DNA for the general transcription factor BTF3.
1990,
Pubmed Zhou,
Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.
2004,
Pubmed Zybailov,
Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae.
2006,
Pubmed
