Identification of inducible nitric oxide synthase binding proteins in human testis: Mammalian co-immunoprecipitation study

Nitric Oxide (NO) is a gaseous molecule involved in a number of physiological processes and is synthesized from L-arginine by a family of nitric oxide synthases (NOSs). Insufficient NO production has been reported to cause hypertension, cardiovascular diseases and impotency. Excessive NO production, produced by inducible nitric oxide synthase (iNOS), on the other hand has been implicated to be responsible for mediating various diseases that include inflammation-based infertility in males. Higher NO levels for longer duration in the cell can target many proteins that interact with it and modify their functions. The molecular mechanism behind the role of NO in male infertility is yet to be revealed. We have been studying the regulation of NO synthesis in tissues particularly human testis. Our strategy was to find which iNOS binding protein(s) (iNOS-bp) are involved in modifying its function thereby controlling the NO production. We started off using previously screened human testis cDNA library clones of possible iNOS-bp by yeast two-hybrid (Y2H) assay. DNA sequencing and BLASTRTM analysis of the putative interacting protein-cDNA samples gave us a bunch of proteins known to be involved in various functions including sperm motility, protein folding & transport, and cancer to name a few. We further carried out a Co-IP using full-length iNOS in a mammalian system, which revealed the novel interaction of Sperm Acrosome Associated Protein-7 (SPACA7) and Retinoblastoma Binding Protein-4 (RbBP4) with iNOS in human testis.