RESUMO
Scorpion venom contains different toxins with multiple biological functions. IMe-AGAP is the first Analgesic-Antitumor like Peptide (AGAP) isolated from Iranian scorpion Mesobuthus eupeus. This peptide is similar to AGAP toxin with high analgesic activity, extracted from Chinese scorpion and inhibits NaV1.8 and NaV1.9 voltage-gated sodium channels involved in the pain pathway. In this study, IMe-AGAP was cloned in a prokaryotic expression vector; expression of toxin in Escherichia coli (E. coli) was assayed and then purified. In in-silico studies, peptide sequence was compared with other scorpion analgesic toxins. The structures of IMe-AGAP and sodium channels were modeled using homology modeling. Structural evaluation and stereo-chemical analysis of modeled structures were performed using RAMPAGE web server Ramachandran plots. Hex Server was used to investigate the interactions between IMe-AGAP and S3-S4 and also S5-S6 segments of NaV1.8 and NaV1.9. Binding energies calculation was used for evaluation of protein docking. Soluble expression of IMe-AGAP in bacteria was investigated by SDS-PAGE analysis. Pure recombinant protein was obtained by Ni-NTA affinity chromatography. The results of three-dimensional structure prediction showed ßαßß topology for the toxin that is similar to the conserved structure of α-toxins. Comparison analysis between IMe-AGAP and AGAP toxins exhibited high similarity in homology modeling. Docking analysis demonstrated that IMe-AGAP can interact with NaV1.8 and NaV1.9 domains involved in pain. According to the results of homology studies and docking, IMe-AGAP might be a novel potential drug for pain treatment.
RESUMO
The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2, and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS, and normal testicular tissues and suggests the possibility of diagnosing the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.