Molecular and structural analysis of central transport channel in complex with Nup93 of nuclear pore complex.

The central transport channel (CTC) of nuclear pore complexes (NPCs) is made up of three nucleoporins Nup62, Nup58 and Nup54. In which manner and capacity, these nucleoporins form the CTC, is not yet clear. We explored the CTC Nups from various species and observed that distinct biochemical characteristics of CTC Nups are evolutionarily conserved. Moreover, comparative biochemical analysis of CTC complexes showed various stoichiometric combinations of Nup62, Nup54 and Nup58 coexisting together. We observed the conserved amino-terminal domain of mammalian Nup93 is crucial for the anchorage of CTC and its localization to NPCs. We could reconstitute and purify mammalian CTC·Nup93 quaternary complex by co-expressing full length or N-terminal domain of Nup93 along with CTC complex. Further, we characterized CTC·Nup93 complex using small angle X-ray scattering and electron microscopy that revealed a "V" shape of CTC·Nup93 complex. Overall, this study demonstrated for the first time evolutionarily conserved plasticity and stoichiometric diversity in CTC Nups.

Structural basis of hypoxic gene regulation by the Rv0081 transcription factor of Mycobacterium tuberculosis.

The transcription factor Rv0081 of Mycobacterium tuberculosis controls hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis (TB) infection. We report here the crystal structure of Rv0081 at 2.9 Å resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. However, unlike other members in this family, Rv0081 has neither a metal-binding domain nor does it possess Cys residues, suggesting an alternate mechanism of gene regulation. Our structural and biochemical analyses suggest the molecular basis for the recognition of self-regulatory DNA sequences and a plausible mechanism of regulation of Rv0081 in the latent phase of TB infection. DATABASE: Structural data are available in the Protein Data Bank under the accession number - 6JMI.

The Nup62 Coiled-Coil Motif Provides Plasticity for Triple-Helix Bundle Formation.

The central transport channel of the vertebrate nuclear pore complex (NPC) consists of nucleoporins: Nup62, Nup54, and Nup58. The coiled-coil domains in α-helical regions of these nucleoporins are thought to be crucial for several protein-protein interactions in the NPC subcomplexes. In this study, we determined the crystal structure of the coiled-coil domain of rat Nup62 fragment (residues 362-425) to 2.4 Å resolution. The crystal structure shows the conserved coiled-coil domain as a parallel three-helix bundle for the Nup62(362-425) fragment. On the basis of our size exclusion chromatography coupled to multiangle light scattering analysis and glutaraldehyde cross-linking experiments, we conclude that the Nup62(362-425) fragment displays dynamic behavior in solution and can also exist in either homodimeric or homotrimeric states. Our comparative analysis of the rat Nup62(362-425) homotrimeric structure with previously reported heterotrimeric structures [rat Nup62(362-425)·Nup54(346-407) and Xenopus Nup62(358-485)·Nup54(315-450)·Nup58(283-406) complexes] demonstrates the structural basis for parallel triple-helix bundle formation for Nup62 with different partners. Moreover, we show that the coiled-coil domain of Nup62 is sufficient for interaction with the coiled-coil domain of rat Exo70, a protein in an exocyst complex. On the basis of these observations, we suggest the plausible chain replacement mechanism that yields to diverse protein assemblies with Nup62. In summary, the coiled-coil motif present in Nup62 imparts the ability to form a homotrimer and heterotrimers either with Nup54 or with Nup54-Nup58 within the NPCs as well as with Exo70 beyond the NPCs. These complexes of Nup62 suggest the crucial role of the coiled-coil motifs in providing plasticity to various modular assemblies.

Functional promoter polymorphisms direct the expression of cystathionine gamma-lyase gene in mouse models of essential hypertension.

Despite the well-known role of cystathionine γ-lyase (Cth) in cardiovascular pathophysiology, transcriptional regulation of Cth remains incompletely understood. Sequencing of the Cth promoter region in mouse models of genetic/essential hypertension (viz. Blood Pressure High [BPH], Blood Pressure Low [BPL] and Blood Pressure Normal [BPN] mice) identified several genetic variations. Transient transfections of BPH/BPL-Cth promoter-reporter plasmids into various cell types revealed higher promoter activity of BPL-Cth than that of BPH-Cth. Corroboratively, endogenous Cth mRNA levels in kidney and liver tissues were also elevated in BPL mice. Computational analysis of the polymorphic Cth promoter region predicted differential binding affinity of c-Rel, HOXA3 and IRF1 with BPL/BPH-Cth promoter domains. Over-expression of c-Rel/HOXA3/IRF1 modulated BPL/BPH-Cth promoter activities in a consistent manner. Gel shift assays using BPH/BPL-Cth-promoter oligonucleotides with/without binding sites for c-Rel/HOXA3/IRF1 displayed formation of specific complexes with c-Rel/HOXA3/IRF1; addition of antibodies to reaction mixtures resulted in supershifts/inhibition of Cth promoter-transcription factor complexes. Furthermore, chromatin immunoprecipitation (ChIP) assays proved differential binding of c-Rel, HOXA3 and IRF1 with the polymorphic promoter region of BPL/BPH-Cth. Tumor necrosis factor-α (TNF-α) reduced the activities of BPL/BPH-Cth promoters to different extents that were further declined by ectopic expression of IRF1; on the other hand, siRNA-mediated down-regulation of IRF1 rescued the TNF-α-mediated suppression of the BPL/BPH-Cth promoter activities. In corroboration, ChIP analysis revealed enhanced binding of IRF1 with BPH/BPL-Cth promoter following TNF-α treatment. BPL/BPH-Cth promoter activity was diminished upon exposure of hepatocytes and cardiomyoblasts to ischemia-like pathological condition due to reduced binding of c-Rel with BPL/BPH-Cth-promoter. Taken together, this study reveals the molecular basis for the differential expression of Cth in mouse models of essential hypertension under basal and pathophysiological conditions.

Post-Transcriptional Regulation of Renalase Gene by miR-29 and miR-146 MicroRNAs: Implications for Cardiometabolic Disorders.

Renalase, a recently identified oxidoreductase, is emerging as a novel regulator of cardiovascular and metabolic disease states. The mechanism of regulation of renalase gene, especially at the post-transcriptional level, is completely unknown. We set out to investigate the possible role of microRNAs in regulation of renalase gene in this study. Computational predictions using multiple algorithms coupled with systematic functional analysis revealed specific interactions of miR-29a/b/c and miR-146a/b with mouse and human renalase 3'-UTR (untranslated region) in cultured cells. Next, we estimated miR-29b and miR-146a, as well as renalase expression, in genetically hypertensive blood pressure high and genetically hypotensive blood pressure low mice. Kidney tissues from blood pressure high mice showed diminished (~1.6- to 1.8-fold) renalase mRNA/protein levels and elevated (~2.2-fold) miR-29b levels as compared to blood pressure low mice. A common single nucleotide polymorphism in human renalase 3'-UTR (C/T; rs10749571) creates a binding site for miR-146a; consistently, miR-146a down-regulated human renalase 3'-UTR/luciferase activity in case of the T allele suggesting its potential role in regulation of renalase in humans. Indeed, genome-wide association studies revealed directionally concordant association of rs10749571 with diastolic blood pressure, glucose and triglyceride levels in large human populations (n ≈ 58,000-96,000 subjects). This study provides evidence for post-transcriptional regulation of renalase gene by miR-29 and miR-146 and has implications for inter-individual variations on cardiometabolic traits.

Transcriptional regulation of the novel monoamine oxidase renalase: Crucial roles of transcription factors Sp1, STAT3, and ZBP89.

Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5'-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions.

Coordinated transcriptional regulation of Hspa1a gene by multiple transcription factors: crucial roles for HSF-1, NF-Y, NF-κB, and CREB.

Although the transcript level of inducible heat shock protein 70.3 (Hsp70.3, also known as Hspa1a) is altered in various disease states, its transcriptional regulation remains incompletely understood. Here, we systematically analyzed the Hspa1a promoter to identify major cis elements and transcription factors that may govern the constitutive/inducible gene expression. Computational analyses coupled with extensive in vitro (promoter-reporter activity and electrophoretic mobility shift assays) and in vivo (chromatin immunoprecipitation assays) revealed interaction of several transcription factors with Hspa1a promoter motifs: HSF-1 (heat shock factor 1) at -114/-97 bp and -788/-777bp, NF-Y (nuclear transcription factor Y) at -73/-58 bp, NF-κB (nuclear factor kappa B) at -133/-124 bp, and CREB (cAMP response element binding protein) at -483/-476 bp. Consistently, siRNA (small interfering RNA)-mediated down-regulation of each of these transcription factors caused substantial reduction of endogenous Hspa1a expression. Heat-shock-induced activation of Hspa1a was coordinately regulated by HSF-1 and NF-Y/NF-κB. The Hspa1a expression was augmented by TNF-α (tumor necrosis factor-alpha) and forskolin in NF-κB and CREB-dependent manners, respectively. NF-κB and CREB also activated Hspa1a transcription in cardiac myoblasts upon exposure to ischemia-like conditions. Taken together, this study discovered previously unknown roles for NF-κB and CREB to regulate Hspa1a expression and a coordinated action by several transcription factors for Hspa1a transactivation under heat-shock/ischemia-like conditions and thereby provided new insights into the mechanism of Hspa1a regulation.

Functional genetic variants of the catecholamine-release-inhibitory peptide catestatin in an Indian population: allele-specific effects on metabolic traits.

Catestatin (CST), a chromogranin A (CHGA)-derived peptide, is a potent inhibitor of catecholamine release from adrenal chromaffin cells and postganglionic sympathetic axons. We re-sequenced the CST region of CHGA in an Indian population (n = 1010) and detected two amino acid substitution variants: G364S and G367V. Synthesized CST variant peptides (viz. CST-Ser-364 and CST-Val-367) were significantly less potent than the wild type peptide (CST-WT) to inhibit nicotine-stimulated catecholamine secretion from PC12 cells. Consistently, the rank-order of blockade of nicotinic acetylcholine receptor (nAChR)-stimulated inward current and intracellular Ca(2+) rise by these peptides in PC12 cells was: CST-WT > CST-Ser-364 > CST-Val-367. Structural analysis by CD spectroscopy coupled with molecular dynamics simulations revealed the following order of α-helical content: CST-WT > CST-Ser-364 > CST-Val-367; docking of CST peptides onto a major human nAChR subtype and molecular dynamics simulations also predicted the above rank order for their binding affinity with nAChR and the extent of occlusion of the receptor pore, providing a mechanistic basis for differential potencies. The G364S polymorphism was in strong linkage disequilibrium with several common CHGA genetic variations. Interestingly, the Ser-364 allele (detected in ∼15% subjects) was strongly associated with profound reduction (up to ∼2.1-fold) in plasma norepinephrine/epinephrine levels consistent with the diminished nAChR desensitization-blocking effect of CST-Ser-364 as compared with CST-WT. Additionally, the Ser-364 allele showed strong associations with elevated levels of plasma triglyceride and glucose levels. In conclusion, a common CHGA variant in an Indian population influences several biochemical parameters relevant to cardiovascular/metabolic disorders.

Molecular interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor.

Catestatin (CST), a chromogranin-A-derived peptide, is a potent endogenous inhibitor of the neuronal nicotinic acetylcholine receptor (nAChR). It exerts an anti-hypertensive effect by acting as a 'physiological brake' on transmitter release into the circulation. However, the mechanism of interaction of CST with nAChR is only partially understood. To unravel molecular interactions of the wild-type human CST (CST-WT) as well as its naturally occurring variants (CST-364S and CST-370L, which have Gly→Ser and Pro→Leu substitutions, respectively) with the human α3ß4 nAChR, we generated a homology-modeled human α3ß4 nAChR structure and solution structures of CST peptides. Docking and molecular dynamics simulations showed that ~90% of interacting residues were within 15 N-terminal residues of CST peptides. The rank order of binding affinity of these peptides with nAChR was: CST-370L>CST-WT>CST-364S; the extent of occlusion of the receptor pore by these peptides was also in the same order. In corroboration with computational predictions, circular dichroism analysis revealed significant differences in global structures of CST peptides (e.g. the order of α-helical content was: CST-370L>CST-WT>CST-364S). Consistently, CST peptides blocked various stages of nAChR signal transduction, such as nicotine- or acetylcholine-evoked inward current, rise in intracellular Ca(2+) and catecholamine secretion in or from neuron-differentiated PC12 cells, in the same rank order. Taken together, this study shows molecular interactions between human CST peptides and human α3ß4 nAChR, and demonstrates that alterations in the CST secondary structure lead to the gain of potency for CST-370L and loss of potency for CST-364S. These findings have implications for understanding the nicotinic cholinergic signaling in humans.

Functional promoter polymorphisms govern differential expression of HMG-CoA reductase gene in mouse models of essential hypertension.

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5'-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure.

Enhancement in the efficiency of polymerase chain reaction by TiO2 nanoparticles: crucial role of enhanced thermal conductivity.

Improvement of the specificity and efficiency of the polymerase chain reaction (PCR) by nanoparticles is an emerging area of research. We observed that TiO(2) nanoparticles of approximately 25 nm diameter caused significant enhancement of PCR efficiency for various types of templates (namely plasmid DNA, genomic DNA and complementary DNA). By a series of experiments, the optimal TiO(2) concentration was determined to be 0.4 nM, which resulted in up to a seven-fold increase in the amount of PCR product. As much as 50% reduction in overall reaction time (by reduction of the number of cycles and the time periods of cycles) was also achieved by utilizing TiO(2) nanoparticles without compromising the PCR yield. Investigations of the mechanism of such PCR enhancement by simulations using the 'Fluent K epsilon turbulent model' provided evidence of faster heat transfer in the presence of TiO(2) nanoparticles. Consistent with these findings, TiO(2) nanoparticles were observed to augment the denaturation of genomic DNA, indicating more efficient thermal conductivity through the reaction buffer. TiO(2) nanoparticle-assisted PCR may be useful for profound reduction of the overall PCR reaction period and for enhanced amplification of DNA amplicons from a variety of samples, including GC-rich templates that are often observed to yield unsatisfactory results.

Chromogranin A: a novel susceptibility gene for essential hypertension.

Chromogranin A (CHGA) is ubiquitously expressed in secretory cells of the endocrine, neuroendocrine, and neuronal tissues. Although this protein has long been known as a marker for neuroendocrine tumors, its role in cardiovascular disease states including essential hypertension (EH) has only recently been recognized. It acts as a prohormone giving rise to bioactive peptides such as vasostatin-I (human CHGA(1-76)) and catestatin (human CHGA(352-372)) that exhibit several cardiovascular regulatory functions. CHGA is over-expressed but catestatin is diminished in EH. Moreover, genetic variants in the promoter, catestatin, and 3'-untranslated regions of the human CHGA gene alter autonomic activity and blood pressure. Consistent with these findings, targeted ablation of this gene causes severe arterial hypertension and ventricular hypertrophy in mice. Transgenic expression of the human CHGA gene or exogenous administration of catestatin restores blood pressure in these mice. Thus, the accumulated evidence establishes CHGA as a novel susceptibility gene for EH.