Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2

Abstract

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1–150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents.

Current Trends in the Analysis of Post-translational Modifications

“…salts, detergents) and more selective than IMAC in many cases, multiply phosphorylated residues may bind with high affinity to the metal oxide, which makes them dramatically difficult to elute. IMAC, therefore, provides improved coverage of multiphosphorylated peptides, and it is also able to enrich full-length phosphoproteins without any prior proteolytic digestion [48][49][50]. Immunoaffinity chromatography is predominantly employed in the enrichment of proteins phosphorylated on the tyrosine residue [51].…”

Pipeline for contraceptive development

“…Male infertility in knock-out mice indicates a critical role for TSSK 1 and 2 in spermiogenesis (78, 79). Progress has been made on isolating a target protein, TSSK 2, and this will facilitate exploration for small molecular drug candidates that could regulate sperm production and function (80). Determining how to use such drugs, so that sperm remain irreversibly inhibited after ejaculation, remains to be demonstrated.…”

Polymorphism of the testis-specific serine/threonine kinase 2 gene and risk of asthenozoospermia in Côte d'Ivoire

“…14,15 In addition, the study conducted by Zhang et al 16 revealed that mutations c.80A>G (rs3747052) and c.774C>T (rs1052756) of the TSSK2 gene could be associated with infertility in men. In addition the study conducted by Xu et al 17 and Shetty et al 13 sho,wed that the inhibition of the activity of the TSSK2 protein for its substrate TSKS could lead to infertility. Indeed, it is involved in the phosphorylation of serine-288 TSKS substrate and SPAG16 protein actively involved in the formation and functioning of the flagellum, essential element of sperm motility.…”

“…10,11 Therefore, TSSK2, a serine / threonine kinase protein that is found specifically in the testicles at the germ cells during spermatogenesis and in mature spermatozoa, is one of the proteins involved in this infertility. 12,13 Previous studies have shown that it plays an important role in the functioning of the spermatozoon. Indeed, it has been reported that mutations in this TSSK2 gene could lead to morphological and functional abnormalities in spermatozoa.…”