HDAC6 interacting-microtubule associated proteins (HMAPs) identified in human sperm

Sperm flagellar axoneme comprises microtubules (MT) and associated machinery and is an integral determinant of sperm motility. Reports from our lab show reduced levels of acetyl α-tubulin, and HDAC6, along with compromised axoneme polymerization in sperm of asthenozoospermic men. These observations prompted us to identify the sperm repertoire of HDAC6-interacting proteins(HIPs) associated with the MTs. HIPs and Microtubule associated protein (MAP) fractions, respectively, were isolated from sperm of normozoospermic individuals, subjected to tandem mass spectrometry(MS) using a bottom-up approach and proteins in the two groups were identified. 1224 and 315 proteins were identified in the respective groups. Seven clusters of HIPs were among the top 20 significant clusters. Proteins were manually curated from these relevant clusters and overlapped with the MAPs dataset which identified 14 HDAC6 interacting proteins to be associated with MTs (HMAPs). On further analysis with MAP analyzer-LZTFL1, RAB7A, AIFM1 demonstrated low specificity toward MT whereas MYH10 and CFAP53 demonstrated high specificity. Among these HMAPs, EEF1A2, MYH10, ANXA1, TUFM, SOD1, and SRSF7 are known to interact with HDAC6 as documented in the BioGRID database. Interaction of CFAP53 with HDAC6 was validated by double immunofluorescence staining and co-immunoprecipitation in rat sperm. LFQ-DDA analysis of these HMAPs, revealed significantly lower abundance of CFAP53 and TUFM with higher abundance of MYH10 in asthenozoospermic men. Their differential expression in men with poor sperm motility as well as enrichment of acetylation on these HMAPs highlights their association with HDAC6 in maintaining axonemal stability/dynamicity and acetylation-deacetylation to the extent required for sperm motility, although interpretation is limited by the small sample size, restricted availability of human sperm for experimental validation, and reliance on in silico acetylation predictions.