Disease relevance of Tnp2

• Cloning of cDNA encoding rat spermatidal protein TP2 and expression in Escherichia coli [1].
• In contrast, transgenic mice bearing only the 525 bp 5'-flanking sequences of rat transition protein 2 gene and 3'-flanking sequences of the simian virus 40 large T antigen showed low levels of transgene expression in testis [2].

High impact information on Tnp2

• The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA) [3].
• We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA) [3].
• Phosphorylation of rat spermatidal protein TP2 by sperm-specific protein kinase A and modulation of its transport into the haploid nucleus [3].
• We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109 [3].
• Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA [4].

Biological context of Tnp2

• Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus [3].
• Identification of two novel zinc finger modules and nuclear localization signal in rat spermatidal protein TP2 by site-directed mutagenesis [4].
• The nucleotide sequence of rat transition protein 2 (TP2) cDNA [5].
• In this study, the 5' regulatory region of rat Tnp2 gene was investigated by primer extension analysis and transgenic mice study [6].

Anatomical context of Tnp2

• Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus [4].
• This testis-specific chromosomal protein is first synthesized during spermatogenesis in pachytene spermatocytes and the entire complement of testis histones is replaced during the midspermatid stage of spermiogenesis by positively charged transition nuclear proteins TP1 and TP2 [7].
• TH2B mRNAs appeared first in preleptotene spermatocytes (PL) on day 12 and in pachytene spermatocytes (PS) on day 18; p19 mRNAs were present in PS from day 18 onward, and TP1 and TP2 mRNAs were detected in round spermatids (RS) from day 32 onward [8].
• The localization of TP2 mRNA to the chromatoid body of step 7 spermatids would be consistent with this organelle being a storage site for long-lived mRNAs utilized later in spermiogenesis [9].

Associations of Tnp2 with chemical compounds

• Phosphorylation experiments with the wild type and different site-specific mutants of TP2 revealed that serine 109 and threonine 101 are the phosphorylation sites [10].
• EDTA altered the circular dichroism spectrum of TP2 and the N-terminal fragment (zinc binding domain) but not that of the C-terminal fragment [11].
• Chemical modification of cysteine by iodoacetic acid, and histidine by diethylpyrocarbonate, resulted in a near complete inhibition of 65Zn-binding to TP2 [11].
• The involvement of cysteine residues of TP2 in coordination with zinc was also suggested by the observation that TP2 could be labeled, in situ, with iodoacetamidofluorescein only after preincubation of spermatid nuclei with EDTA [12].
• Upon induction with 1 mM IPTG, there was a low level of expression of TP2 which could be recovered in the soluble form [1].

Other interactions of Tnp2

• Spermatidal transition protein, TP2, was purified from rat testes by Hg-affinity chromatography [11].

Analytical, diagnostic and therapeutic context of Tnp2

• We have carried out a detailed site-directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc [4].
• Circular dichroism spectroscopy studies of TP2 (zinc pre-incubated) and its V8 protease-derived polypeptide fragments revealed that the N-terminal fragment has a Type I-beta-turn spectrum, while the C-terminal fragment has a small but significant alpha-helical structure [11].
• TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments [13].
• We report here cloning of complementary DNA encoding rat TP2 by the RT-PCR method [1].
• Recombinant TP2 was purified from the soluble extract of E. coli using nickel-agarose and heparin-agarose chromatography and was shown to be identical to native rat TP2 as revealed by immunoblotting with anti-rat TP2 antibodies and radioactive 65Zn-blotting [1].

References