Sperm phosphoproteomics: historical perspectives and current methodologies

Porambo, James R; Salicioni, Ana M.; Visconti, Pablo E.; Platt, Mark

doi:10.1586/epr.12.41

Cited by 35 publications

(28 citation statements)

References 119 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When enrichment efficiency was compared, the percentage of acetylated peptides over total peptides ranged from ϳ12 to 31%, which was higher than that of polyclonal antibodies (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)see Ref. 32).…”

Section: Discussionmentioning

confidence: 89%

See 1 more Smart Citation

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)

Diao

Wang

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pananti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions. Molecular & Cellular

show abstract

Section: Discussionmentioning

confidence: 89%

“…The importance of phosphorylation, an essential PTM in sperm differentiation, maturation, and fertilization, has been well established (16,17). Acetylation as a broad and abundant PTM comparable with phosphorylation (18), however, has not been well analyzed in human sperm.…”

mentioning

confidence: 99%

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)

Diao

Wang

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…Enhanced chemioluminescence ECL Plus Kit (GE Healthcare) was used for detection. Tyrosine phosphorylated hexokinase served as a loading control (Porambo et al, 2012;Visconti et al, 1995a). When necessary, PVDF membranes were stripped at 65°C for 20 min in 2% SDS, 0.74% β-mercaptoethanol, 62.5 mM Tris, pH 6.5, and then washed six times for 5 min each in T-TBS prior to re-probing.…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm

et al. 2016

View full text Add to dashboard Cite

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2 −/− mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro.

show abstract

“…Moreover, a recent study characterized the testicular phosphoproteome in the perfluorododecanoic acid (PFDoA)-exposed rats, and demonstrated the importance of MAPK pathway and CDC2 protein phosphorylation in the toxicity of PFDoA (19). Taken together, despite the fact that a number of studies have been carried out (18), our understanding of the testicular phosphoproteome is still limited, and more effort needs to be expended on this area.…”

mentioning

confidence: 99%

Systematic Analysis of the Phosphoproteome and Kinase-substrate Networks in the Mouse Testis

Lin

Liu

Wang

et al. 2014

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Spermatogenesis is a complex process closely associated with the phosphorylation-orchestrated cell cycle. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3955 phosphoproteins. Although only approximately half of the phosphorylation sites enriched by IMAC were also captured by TiO 2 , both the phosphoprotein data sets identified by the two methods significantly enriched the functional annotation of spermatogenesis. Thus, the phosphoproteome profiled in this study is a highly useful snapshot of the phosphorylation events in spermatogenesis. To further understand phosphoregulation in the testis, the site-specific kinasesubstrate relations were computationally predicted for reconstructing kinase-substrate phosphorylation networks. A core sub-kinase-substrate phosphorylation networks among the spermatogenesis-related proteins was retrieved and analyzed to explore the phosphoregulation during spermatogenesis. Moreover, network-based analyses demonstrated that a number of protein kinases such as MAPKs, CDK2, and CDC2 with statistically more sitespecific kinase-substrate relations might have significantly higher activities and play an essential role in spermatogenesis, and the predictions were consistent with previous studies on the regulatory roles of these kinases. In particular, the analyses proposed that the activities of POLO-like kinases (PLKs) might be dramatically higher, while the prediction was experimentally validated by detecting and comparing the phosphorylation levels of pT210, an indicator of PLK1 activation, in testis and other tissues. Further experiments showed that the inhibition of POLO-like kinases decreases cell proliferation by inducing G2/M cell cycle arrest. Taken together, this systematic study provides a global landscape of phosphoregulation in the testis, and should prove to be of value in future studies of spermatogenesis. Molecular & Cellular Proteomics 13:

show abstract

Sperm phosphoproteomics: historical perspectives and current methodologies

Cited by 35 publications

References 119 publications

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm

Systematic Analysis of the Phosphoproteome and Kinase-substrate Networks in the Mouse Testis

Contact Info

Product

Resources

About