Revelation of endogenously bound Fe(2+) ions in the crystal structure of ferritin from Escherichia coli.

Ferritin is an iron regulatory protein. It is responsible for storage and detoxification of excess iron thereby it regulates iron level in the body. Here we report the crystal structure of ferritin with two endogenously expressed Fe atoms binding in both the sites. The protein was purified and characterized by MALDI-TOF and N-terminal amino acid sequencing. The crystal belongs to I4 space group and it diffracted up to 2.5Å. The structural analysis suggested that it crystallizes as hexamer and confirmed that it happened to be the first report of endogenously expressed Fe ions incorporated in both the A and B sites, situated in between the helices.

Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans.

RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP-l-histidine-Mg(2+)) and EDTA (0.5 M) treated ternary complex (HutP-l-histidine-Mg(2+)), solved at 1.9 Å and 2.5 Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5 M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.

Histone variants enriched in oocytes enhance reprogramming to induced pluripotent stem cells.

Expression of Oct3/4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Somatic cell nuclear transfer (SCNT) can also be used for reprogramming, suggesting that factors present in oocytes could potentially augment OSKM-mediated induction of pluripotency. Here, we report that two histone variants, TH2A and TH2B, which are highly expressed in oocytes and contribute to activation of the paternal genome after fertilization, enhance OSKM-dependent generation of iPSCs and can induce reprogramming with Klf4 and Oct3/4 alone. TH2A and TH2B are enriched on the X chromosome during the reprogramming process, and their expression in somatic cells increases the DNase I sensitivity of chromatin. In addition, Xist deficiency, which was reported to enhance SCNT reprogramming efficiency, stimulates iPSC generation using TH2A/TH2B in conjunction with OSKM, but not OSKM alone. Thus, TH2A/TH2B may enhance reprogramming by introducing processes that normally operate in zygotes and during SCNT.

Design of a novel nucleoside analog as potent inhibitor of the NAD dependent deacetylase, SIRT2.

Sirtuins (class III histone deacetylase) are evolutionarily conserved NAD(+)-dependent enzymes that catalyze the deacetylation of acetyl-lysine residues of histones and other target proteins. Because of their associations in various pathophysiological conditions, the identification of small molecule modulators has been of significant interest. In the present study, virtual screening was carried out with NCI Diversity Set II using crystal structure of hSIRT2 (PDB ID: 1J8F) as a model for the docking procedure to find potential compounds, which were then subjected to experimental tests for their in vitro SIRT2 inhibitory activity. One of the 40 compounds tested, NSC671136 (IUPAC name: 6-Acetyl-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-5-yl 2,4-dichlorobenzoate) has structurally unique scaffold, showed strong inhibitory activity towards SIRT2 with IC(50) of ~8.7 µM and to a lesser extent on SIRT1 activity. The reported compound is substantially potent compared to the published SIRT2 inhibitors and serves as an excellent base for future lead development.