Mutation in angiotensin II type 1 receptor disrupts its binding to angiotensin II leading to hypotension: An insight into hydrogen bonding patterns

To understand the role of angiotensin II type 1 receptor gene (AGTR1) gene products in relation to hypotension we have analyzed the single nucleotide polymorphisms (SNPs) associated with this gene. This can help us to understand the genetic variations that can alter the function of the gene products. In this present study, we report the polymorphic variant associated with AGTR1 and its weak interaction with angiotensin II (AngII) which leads to hypotension. Out of 1318 SNPs, six are found to be non-synonymous, of which rs1064533 shows significant damaging effect. A missense mutation (T1255G), i.e., from thymine to guanine for rs1064533 in AGTR1 gene results in amino acid substitution from cysteine (Cys) to tryptophan (Trp) in the receptor protein. A strong hydrogen bond exists between Cys289 of native AGTR1 protein and glutamine 167 of AngII. Interestingly, it is replaced by a weak hydrogen bond in the mutant protein between Trp289 (mutant residue) and serine 340. Such a substitution from small, hydrophilic to bulky, hydrophobic residue in AGTR1 protein results in reduced binding affinity of the receptor protein with AngII, leading to hypotension. The results presented from this in silico study will open up new prospect for genetic analysis of AGTR1 gene and will be beneficial to the researchers for understanding the role played by AGTR1 gene in hypotension disease.     

Propranolol has direct antithyroid activity: Inhibition of iodide transport in cultured thyroid follicles

The effect of propranolol on the process of thyroid hormone formation was studied in a physiological culture system. Porcine thyroid follicles were preincubated with propranolol for 24 h. Iodide transport, iodine organification, and de novo thyroid hormone formation were measured by incubating these follicles with the mixture of carrier-free 0·1 μCi Na ¹²⁵I and 50 nM NaI for 2 to 6 h at 37°C. A concentration of propranolol greater than 100 μM inhibited iodide transport in a dose-dependent manner; this inhibition was non-competitive with iodide and independent of thyrotropin (TSH). Reduced iodine organification and thyroid hormone formation was seen with 150 μM propranolol or greater. The inhibitory action of propranolol was not caused by beta-blocking activity, since D -propranolol (devoid of beta-blocking activity) inhibited iodide transport, and other beta-blockers (metoprolol, atenolol, and labetalol) did not inhibit iodide transport. The inhibition of iodide transport was most likely caused by membrane stabilizing activity since quinidine, which possess the same membrane stabilizing activity as propranolol, also inhibited iodide transport. TSH-mediated cAMP generation and Na ⁺K⁺ ATPase activity, membrane functions for iodide transport, were unaffected by propranolol. Our study has shown, for the first time, that propranolol has a direct antithyroid action, namely inhibition of iodide transport in the intact thyroid follicle.     

Thermal stability of the K+ channel tetramer: cation interactions and the conserved threonine residue at the innermost site (S4) of the KcsA selectivity filter

The selectivity filter of most K+ channels contains a highly conserved Thr residue that uniquely forms the S4 binding site for K+ by dual coordination with the backbone carbonyl oxygen and side chain hydroxyl of the same residue. This study examines the effect of mutations of Thr75 in the S4 site of theKcsA K+ channel on the cation dependence of the thermal stability of the tetramer, a phenomenon that reflects the structural role of cations in the filter. Conservative mutations of Thr75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity ofK+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by approximately 10(3)- or approximately 10(4)-fold for Ala and Cys substitution, respectively,consistent with the known preference of the S4 site for Ba2+. In contrast, substitution of Ala or Cys at T75 anomalously enhances the ability of Na+ to stabilize the tetramer, suggesting that the native Thr residue at S4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature orCu2+ cation catalyzes formation of covalent dimers of the T75C mutant of KcsA via formation of disulfide bonds between Cys residues of adjacent subunits. Thiophilic cations such as Hg2+ and Ag+ specifically protect the T75C tetramer against heat-induced dimer formation, demonstrating the contribution of cation interactions to tetramer stability in a channel with a non-native S4 site engineered to bind foreign cations.     

Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor

Agonist-stimulated beta(2)-adrenergic receptor (beta(2)AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the beta(2)AR in HEK-293 cells. Moreover, siRNA that down-regulates Nedd4 expression inhibited beta(2)AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, beta(2)AR as well as beta-arrestin2, the endocytic and signaling adaptor for the beta(2)AR, interact robustly with Nedd4 upon agonist stimulation. However, beta(2)AR-Nedd4 interaction is ablated when beta-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for beta-arrestin2 in mediating beta(2)AR ubiquitination. Notably, beta-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in beta(2)AR trafficking. Collectively, our findings indicate that the degradative fate of the beta(2)AR in the lysosomal compartments is dependent upon beta-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination.     

Suppression of DeltabipA phenotypes in Escherichia coli by abolishment of pseudouridylation at specific sites on the 23S rRNA

The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by DeltabipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.     

Pathogen proliferation governs the magnitude but compromises the function of CD8 T cells

CD8+ T cell memory is critical for protection against many intracellular pathogens. However, it is not clear how pathogen virulence influences the development and function of CD8+ T cells. Salmonella typhimurium (ST) is an intracellular bacterium that causes rapid fatality in susceptible mice and chronic infection in resistant strains. We have constructed recombinant mutants of ST, expressing the same immunodominant Ag OVA, but defective in various key virulence genes. We show that the magnitude of CD8+ T cell response correlates directly to the intracellular proliferation of ST. Wild-type ST displayed efficient intracellular proliferation and induced increased numbers of OVA-specific CD8+ T cells upon infection in mice. In contrast, mutants with defective Salmonella pathogenicity island II genes displayed poor intracellular proliferation and induced reduced numbers of OVA-specific CD8+ T cells. However, when functionality of the CD8+ T cell response was measured, mutants of ST induced a more functional response compared with the wild-type ST. Infection with wild-type ST, in contrast to mutants defective in pathogenicity island II genes, induced the generation of mainly effector-memory CD8+ T cells that expressed little IL-2, failed to mediate efficient cytotoxicity, and proliferated poorly in response to Ag challenge in vivo. Taken together, these results indicate that pathogens that proliferate rapidly and chronically in vivo may evoke functionally inferior memory CD8+ T cells which may promote the survival of the pathogen.     

A-Kinase Anchoring Protein Targeting of Protein Kinase A and Regulation of HERG Channels

Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K(+) current (I (Kr)), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I (Kr) by cAMP.