HSPA2 Emerges as a Key Biomarker: Insights from Global Lysine Acetylproteomic Profiling in Idiopathic Male Infertility.

Abstract

Spermatozoa are highly specialized cells, and any alterations in their protein profiles may affect their function and fertilizing ability. In spermatozoa, which are transcriptionally and translationally inactive, molecular chaperones particularly heat shock proteins play crucial roles in maintaining redox balance and preserving protein integrity. Post-translational modifications, particularly lysine acetylation, influence chaperone function and are lately being recognized in the pathophysiology of male infertility. To assess the impact of lysine acetylation on sperm chaperone proteins in idiopathic infertile patients (IIP) compared to fertile donors (FD), we performed immunoprecipitation coupled with LC-MS/MS analysis of lysine acetylated sperm proteins from both groups. Proteomic analysis revealed 2,988 acetylated proteins, comprising 26 chaperone proteins that were differentially expressed with four upregulated and nine downregulated in the IIP group. Functional analyses demonstrated enrichment of these proteins in protein folding, spermatogenesis, and response to oxidative stress. CytoHubba analysis reported, key HSP70 family members, HSPA2, HSPA4, and HSPA1A as central hub proteins in protein-protein interaction networks. STRING and IPA network analyses further highlighted the central regulatory roles of these chaperones, with HSPA2 emerging as a key hub protein based on friendship analysis. Western blot validation revealed hypoacetylation and downregulation of HSPA2 in spermatozoa from the IIP group, accompanied by elevated levels of 4-Hydroxynonenal (4-HNE), indicating a link between redox imbalance and altered lysine acetylation in chaperone proteins. Additionally, intense aniline blue staining of sperm nuclei in the IIP group suggested aberrant spermiogenesis. Considering HSPA2's well-documented involvement in sperm maturation and oocyte recognition, its diminished acetylation and expression may not only act as a potential biomarker but also contribute mechanistically to the development of idiopathic male infertility. This study underscores the significance of lysine acetylation in HSPA2 in regulating chaperone function and highlights its diagnostic and therapeutic potential in unexplained male infertility.