PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

Recent research has emphasized the significance of testis-specific serine proteases in regulating various aspects of sperm maturation and function. Among them, serine protease 55 (PRS55) plays an important role in the energy metabolism of sperm and is essential for male fertility in mice. A recent case study further suggests its potential importance to human fertility. However, the underlying molecular mechanism by which PRS55 influences sperm function are still not well understood. The present study aims to investigate these mechanisms further.

In this study, we found impaired mitochondrial function in Prss55^-/- testes and sperm with low ATP production, and decreased mitochondrial membrane potential in sperm. We then validated that PRSS55 is mainly localized in mitochondria by immunofluorescence staining of sperm and transfected NIH-3T3 cells and immunoblotting of testis and transfected HEK293 cells. In HEK293 cells, overexpression of PRSS55 improved mitochondrial function in vitro, as increased ATP production and NAD+ /NADH ratio. By proteomics, we identified 1593 and 711 differentially expressed proteins (DEPs, fold change > 1.5 and corrected P ≤ 0.05) in PRSS55-enriched testicular cells and sperm between wt and Prss55^-/- mice, respectively. Functional annotation revealed that these DEPs were mainly associated with energy metabolic pathways, especially branched-chain amino acid (BCAA) metabolism and oxidative phosphorylation process. In subsequent metabolomic analysis, we observed significant accumulations of BCAAs (valine, leucine and isoleucine) in Prss55^-/- testes. Using LC-MS/MS and Co-IP assays, we discovered and validated that PRSS55 was interacting with two key molecules in BCAA metabolism as branched-chain alpha-ketoacid dehydrogenase kinase (BCKDK) and its substrate mitochondrial branched-chain ketoacid dehydrogenase E1α (BCKDHA).

Our study demonstrates that PRSS55 interacts with BCKDK and BCKDHA, and regulates BCAA metabolism and energy homeostasis, thereby facilitating sperm migration. Our study provides a biological rationale for PRSS55 as a potential therapeutic target for the treatment of male infertility in clinical.