Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa

Vigodner, Margarita; Shrivastava, Vibha; Gutstein, Leah Elisheva; Schneider, Jordana; Nieves, Edward; Goldstein, Marc; Feliciano, M.; Callaway, Myrasol

doi:10.1093/humrep/des317

Cited by 60 publications

(67 citation statements)

References 33 publications

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“…In addition, we found that when sumoylation is analysed using a short-time (4 min) permeabilisation protocol by an immunofluorescence/flow cytometric method, it is mostly detected in live sperm and the percentage of SUMO1-positive live sperm negatively correlates with progressive and total motility in asthenospermic men , indicating that sumoylation, when evaluated with such a protocol, may be a marker of immotile live sperm. However, when a long-time permeabilisation protocol was employed, a high percentage of sperm was found to express SUMO1 , in line with recent results (Vigodner et al 2013) obtained with a different permeabilisation protocol where a relation between excessive sumoylation and abnormal sperm morphology was unmasked. Overall, these results indicate that sumoylation could be necessary (being present in most sperm) and, at the same time, deleterious (marking immotile and morphologically abnormal sperm) for human sperm functions depending on its extension, its localisation within the cell and, likely, on the specific proteins that are being SUMO-modified.…”

Section: Introductionsupporting

confidence: 87%

“…Therefore, post-translational modifications represent the main way for sperm to acquire their functionality (Blaquier et al 1988a,b, Ross et al 1990). Among post-translational modifications occurring in mature sperm (Muratori et al 2011), protein sumoylation has been recently described , Vigodner et al 2013. Sumoylation consists in the attachment of small ubiquitin-like modifier (SUMO) peptides via its C-terminal glycine residue to the lysine residues of the protein targets, mediated by E1, E2 and E3 enzymes (Geiss-Friedlander & Melchior 2007).…”

Section: Introductionmentioning

confidence: 99%

“…In such a scenario, identification of targets will give insights in understanding the functions of the SUMO pathway in sperm. Although several SUMO2/3-ylated proteins were identified by mass spectrometry (Vigodner et al 2013), the role of sumoylation of these proteins in sperm functions remains undefined.…”

Section: Introductionmentioning

confidence: 99%

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SUMO1 in human sperm: new targets, role in motility and morphology and relationship with DNA damage

et al. 2014

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In studies carried out previously, we demonstrated that small ubiquitin-like modifier 1 (SUMO1) is associated with poor sperm motility when evaluated with a protocol that reveals mostly SUMO1-ylated live sperm. Recently, with another protocol, it has been demonstrated that SUMO is expressed in most sperm and is related to poor morphology and motility, suggesting that sumoylation may have multiple roles depending on its localisation and targets. We show herein, by confocal microscopy and co-immunoprecipitation, that dynaminrelated protein 1 (DRP1), Ran GTPase-activating protein 1 (RanGAP1) and Topoisomerase IIa, SUMO1 targets in somatic and/or germ cells, are SUMO1-ylated in mature human spermatozoa. DRP1 co-localises with SUMO1 in the mid-piece, whereas RanGAP1 and Topoisomerase IIa in the post-acrosomal region of the head. Both SUMO1 expression and co-localisation with the three proteins were significantly higher in morphologically abnormal sperm, suggesting that sumoylation represents a marker of defective sperm. DRP1 sumoylation at the mid-piece level was higher in the sperm of asthenospermic men. As in somatic cells, DRP1 sumoylation is associated with mitochondrial alterations, this protein may represent the link between SUMO and poor motility. As SUMO pathways are involved in responses to DNA damage, another aim of our study was to investigate the relationship between sumoylation and sperm DNA fragmentation (SDF). By flow cytometry, we demonstrated that SUMO1-ylation and SDF are correlated (rZ0.4, P!0.02, nZ37) and most sumoylated sperm shows DNA damage in co-localisation analysis. When SDF was induced by stressful conditions (freezing and thawing and oxidative stress), SUMO1-ylation increased. Following freezing and thawing, SUMO1-Topoisomerase IIa co-localisation and co-immunoprecipitation increased, suggesting an involvement in the formation/repair of DNA breakage.

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Section: Introductionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

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SUMO1 in human sperm: new targets, role in motility and morphology and relationship with DNA damage

et al. 2014

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“…Although it is known that sumoylation helps maintain overall metabolic homeostasis and facilitates cellular stress response through the regulation and/or adaptation of the most fundamental metabolic processes, the exact molecular mechanism is still unknown (44). Several SUMO substrates, such as PKM2, G6PD, LDHC, PGK1, and GBE1, identified in this study and others are well-known metabolic enzymes (15,(45)(46)(47). Investigating how sumoylation regulates their functions will undoubtedly improve our understanding of metabolic regulation.…”

Section: + ------+ + + + --+ ------------+ ------------+ ------------mentioning

confidence: 68%

Identification of Small Ubiquitin-like Modifier Substrates with Diverse Functions Using the Xenopus Egg Extract System

Aslanian

Sun

et al. 2014

Molecular & Cellular Proteomics

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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 SUMOconjugated 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. Molecular & Cellular Proteomics 13: 10.1074/ mcp.M113.035626, 1659-1675, 2014. Post-translational modification by small ubiquitin-like modifier (SUMO)1 is highly conserved from yeast to human. There are three well-characterized SUMO proteins in human: SUMO1, 2, and 3. SUMO2 and SUMO3 share about 92% identity and have no apparent functional differences. However, the identity between SUMO2/3 and SUMO1 is only 45%. SUMO1 has different dynamics and a distinct profile of target proteins from SUMO2/3, and some proteins with SUMOinteracting motifs (SIMs) distinguish SUMO1 from SUMO2/3. Therefore, protein substrates modified by different SUMO proteins may exert different functions (1).Sumoylation is a multi-step enzymatic process. The SUMO precursor protein is initially processed at the C terminus by SUMO-specific proteases (SENPs) to generate mature SUMO with a C-terminal Gly-Gly motif. Then, an ATP-dependent activating E1 enzyme (a heterodimer of SAE1 and SAE2) and a conjugating enzyme E2 (UBC9) link sequentially to SUMO by forming a thioester bond between SUMO's C-terminal Gly and Cys residues in the active sites of these enzymes. In an E3 ligase dependent or independent manner, SUMO is conjugated through the C-terminal Gly-Gly motif onto the -amino group of specific Lys residues of substrates forming an isopeptide bond. The substrates can be modified by a single SUMO, multiple SUMOs at several Lys r...

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“…Finally, Vigodner et al localized areas of increased SUMOylation (small ubiquitin-like modifiers) in specific regions of morphologically abnormal human sperm. Through MS they managed to identify 55 SUMOylated proteins, such as HSPs, proteins involved in metabolism, sperm maturation -some of which were sperm-specific-, including HSPA2, A-kinase anchor proteins 3 and 4, Llactate dehydrogenase C, valosin-containing protein and seminogelins (66). Despite the increasing number of studies on spermatozoa, the majority of proteomic analyses have been conducted on seminal plasma (SP), which comprises 90% of the semen and is the supernatant remaining after centrifugation of the semen (67).…”

Section: Proteomics and Spermmentioning

confidence: 99%

The Use of Proteomics in Assisted Reproduction

Kosteria

Anagnostopoulos

Kanaka‐Gantenbein

et al. 2017

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Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa

Abstract: Numerous proteins are modified by sumoylation in human sperm; excessive sumoylation is a marker of defective spermatozoa.

Cited by 60 publications

References 33 publications

SUMO1 in human sperm: new targets, role in motility and morphology and relationship with DNA damage

SUMO1 in human sperm: new targets, role in motility and morphology and relationship with DNA damage

Identification of Small Ubiquitin-like Modifier Substrates with Diverse Functions Using the Xenopus Egg Extract System

The Use of Proteomics in Assisted Reproduction

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