Abstract
Protein ubiquitination, a major and conserved post-translational modification, is known to play a critical regulatory role in many biological processes in eukaryotes. Although several ubiquitinated proteins have been found in buffalo (Bubalus bubalis) testis in our previous studies, large-scale profiling of buffalo testis ubiquitome has not been reported to date. In the present study, we first identified a global profiling of lysine ubiquitination of adult buffalo testis using a highly sensitive LC-MS/MS coupled with immune-affinity enrichment of ubiquitinated peptides. In total, 422 lysine ubiquitination sites were identified in 262 proteins in adult buffalo testis tissue. Bioinformatics analysis showed that the ubiquitinated proteins are involved in a variety of biological processes and diverse subcellular localizations. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein interaction network analysis indicated that proteasome, glycolysis/gluconeogenesis and gap junction pathways are modulated by protein ubiquitination in testis. Besides, 44 ubiquitinated proteins may involve in spermatogenesis according to the SpermatogenesisOnline database, of which, the ubiquitination of HSPA2 and UCHL1 were confirmed by Immunoprecipitation (IP)/Western blot analysis. Taken together, these data provide a global view of ubiquitome in buffalo testis for the first time, and serve as an important resource for exploring the physiological role especially spermatogenesis of lysine ubiquitination in testis in mammals.
Highlights
Ubiquitin (Ub) is a small 76-amino-acid protein that is highly conserved in eukaryotic organisms
In order to study the roles of ubiquitinated proteins in buffalo testis, we performed proteome-wide analysis of lysine ubiquitination sites and proteins in normal adult buffalo testis
It is noteworthy that 4.2% (11) of proteins contained five or more lysine ubiquitination sites and f had at least nine sites (Supplementary Table S4)
Summary
Ubiquitin (Ub) is a small 76-amino-acid protein that is highly conserved in eukaryotic organisms. It is conjugated to the ε-amino group of Lysines (Lys) and always found in both cytosol and nucleus of eukaryotic cells [1]. The ubiquitin–proteasome system (UPS) is facilitated to protein degradation and consisted of three major enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligase enzymes (E3) [2]. The testis is an important sexual organ in male mammals. Testis is the site of spermatogenesis, a complicated and dynamic process in which the male germ cell metamorphoses into mature spermatozoa. Three major events including spermatocytogenesis, meiosis, as well as spermiogenesis occurred in the seminiferous tubules. The spermatogonia must undergo meiosis and the round spermatids have to abandon redundant cytoplasm materials, in other words, many proteins and License 4.0 (CC BY)
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