TRPV3 mutants causing <i>Olmsted Syndrome</i> induce impaired cell adhesion and nonfunctional lysosomes

<p>TRPV3 is a non-selective cationic channel and is important for several physiological functions. It can be activated by physiological temperature and selective endogenous and exogenous compounds. TRPV3 is one of the key ion channel involved in Ca²⁺-signaling in keratinocyte and thus involved in skin-related functions. Recently, naturally occurring mutations in TRPV3, namely G573A, G573S, G573C and W692G have been detected which are linked with the development of pathophysiological conditions such as <i>Olmsted Syndrome</i> (OS) and other skin disorders. Our qualitative and quantitative data suggests that these naturally occurring TRPV3 mutants are mainly restricted in the ER. Expression of OS-mutants cause impaired vesicular trafficking resulting reduced surface localization of these mutants and other membrane proteins too. OS-mutants also cause reduced cell adhesion, altered distribution and less number of lysosomes. Our data confirms that TRPV3 is a lysosomal protein suggesting that <i>Olmsted Syndrome</i> is a lysosomal disorder. These findings may have a broad implication in the context of keratinocyte functions, skin-degeneration and in skin-cancer.</p

Analysis of conservation of different domains, motifs and interacting sites in TRPV1.

<p>The lower value indicates more conservation and higher value indicates less conservation. Different regions of the TRPV1 are indicated by different colors. Ank: Ankyrin repeat region, TM: Transmembrane region, L: Loop region, CBD: Cholesterol-binding domain, P-loop; Pore loop, TRP-box: Signature motif for TRP box, TBS-1: Tubulin binding sequence 1, TBS-2: Tubulin binding sequence 2, TAD-1: Tetrameric assembly domain 1, TAD-2: Tetrameric assembly domain 2, Cam: Calmodulin binding region, NT: N-terminal cytoplasmic domain of TRPV1, MID: Middle portion of TRPV1, CT: C-terminus of TRPV1, FL: Full length TRPV1, Histone 4: Histone 4. All values are significant (P<0.0001, Kuskal-Wallis test).</p

Bayesian phylogeny of TRPV1 illustrates that there is a single copy of this gene is conserved across different vertebrates.

<p>Bayesian phylogenetic tree of TRPV1 proteins from mammals (red) birds-reptiles (yellow) and fishes (green) was generated using MrBayes 3.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031448#pone.0031448-Ronquist1" target="_blank">[52]</a>. Percentage posterior probabilities are marked at the node of the branches while mean branch length is marked in decimal on the respective branch. Putative TRPV like gene (JGI accession id e_gw1.02q.75.1) from “Ciona intestinalis” served as out-group in this phylogenetic tree.</p

Description of different domains and motifs.

<p>Description of different domains and motifs.</p

Distribution of positively charged residues within the TBS-1 and TBS-2 are evolutionary conserved.

<p><b>a.</b> The distribution of positively charged residues located within TBS-1 and TBS-2 of human TRPV1 are represented in a 360° circular wheel. <b>b.</b> The helix formed by TBS-1 and TBS-2 from different species are superimposed and the distribution of positively charged residues are represented in a 360° circular wheel. The positions are marked by alphabets and A indicated the position of first positively charged residues in the helical wheel that has been placed at 0° angle. The distribution of positively charged residues in both TBS-1 and TBS-2 remain much conserved. Residues - lysine (K), arginine (R) and histidine (H) are represented by red green, and the indigo dots, respectively.</p

The primary sequence of TBS-1 and TBS-2 are semi-conserved throughout the evolution.

<p>The conservation of the basic amino acids is indicated.</p

Molecular evolution of TRPV1.

<p>Conservation analysis with TRPV1 in comparison to histone H4 and cytochrome C.</p

Upregulation, Functional Association, and Correlated Expressions of TRPV1 and TRPA1 During Telmisartan-Driven Immunosuppression of T Cells

TRPV1 and TRPA1, are known to be functionally expressed in T cells, where these two channels differentially regulate effector immune responses. Telmisartan (TM), an anti-hypertension drug, has been recently repurposed to suppress various inflammatory responses. However, the possible involvement of TRP channels during TM-driven suppression of T cells responses has not been explored yet. In this study, we investigated the potential role of TRPV1 and TRPA1 during TM-driven immunosuppression of T cells in vitro. We observed a significant elevation of both TRPV1 and TRPA1 during TM-induced immunosuppression of T cells.We found that TRPA1 activation-driven suppression of T cell activation and effector cytokine responses during TM treatment is partially, yet significantly overridden by TRPV1 activation. Moreover, the expressions of TRPV1 and TRPA1 were highly correlated in various conditions of T cell. Mechanistically, it might be suggested that TRPV1 and TRPA1 are differentially involved in regulating T cell activation despite the co-elevation of both these TRP channels’ expressions in the presence of TM. T cell activation was delineated by CD69 and CD25 expressions along with the effector cytokine levels (IFN-γ and TNF) in TM-driven suppression of T cell. These findings could have broad implications for designing possible future immunotherapeutic strategies, especially in the repurposing of TM for T cell-TRP-directed immune disorders.</p

Data_Sheet_1_Antibacterial Efficacy of Polysaccharide Capped Silver Nanoparticles Is Not Compromised by AcrAB-TolC Efflux Pump.pdf

<p>Antibacterial therapy is of paramount importance in treatment of several acute and chronic infectious diseases caused by pathogens. Over the years extensive use and misuse of antimicrobial agents has led to emergence of multidrug resistant (MDR) and extensive drug resistant (XDR) pathogens. This drastic escalation in resistant phenotype has limited the efficacy of available therapeutic options. Thus, the need of the hour is to look for alternative therapeutic approaches to mitigate healthcare concerns caused due to MDR bacterial infections. Nanoparticles have gathered much attention as potential candidates for antibacterial therapy. Equipped with advantages of, wide spectrum bactericidal activity at very low dosage, inhibitor of biofilm formation and ease of permeability, nanoparticles have been considered as leading therapeutic candidates to curtail infections resulting from MDR bacteria. However, substrate non-specificity of efflux pumps, particularly those belonging to resistance nodulation division super family, have been reported to reduce efficacy of many potent antibacterial therapeutic drugs. Previously, we had reported antibacterial activity of polysaccharide-capped silver nanoparticles (AgNPs) toward MDR bacteria. We showed that AgNPs inhibits biofilm formation and alters expression of cytoskeletal proteins FtsZ and FtsA, with minimal cytotoxicity toward mammalian cells. In the present study, we report no reduction in antibacterial efficacy of silver nanoparticles in presence of AcrAB-TolC efflux pump proteins. Antibacterial tests were performed according to CLSI macrobroth dilution method, which revealed that both silver nanoparticles exhibited bactericidal activity at very low concentrations. Further, immunoblotting results indicated that both the nanoparticles modulate the transporter AcrB protein expression. However, expression of the membrane fusion protein AcrA did show a significant increase after exposure to AgNPs. Our results indicate that both silver nanoparticles are effective in eliminating MDR Enterobacter cloacae isolates and their action was not inhibited by AcrAB-TolC efflux protein expression. As such, the above nanoparticles have strong potential to be used as effective and alternate therapeutic candidates to combat MDR gram-negative Enterobacterial pathogens.</p

Image2_TRPV1 channel in spermatozoa is a molecular target for ROS-mediated sperm dysfunction and differentially expressed in both natural and ART pregnancy failure.tif

Bi-directional crosstalk between Ca2+ signaling and ROS modulates physiological processes as a part of a regulatory circuit including sperm function. The role of transient receptor potential vanilloid 1 (TRPV1) in this regard cannot be undermined. This is the first report demonstrating the Ca2+-sensitive TRPV1 channel to be under-expressed in spermatozoa of subfertile men, idiopathic infertile men, and normozoospermic infertile males with high ROS (idiopathic infertility and unilateral varicocele). To study the effect of TRPV1 in determining the fertility outcome, we compared the expression profile of TRPV1 in spermatozoa of male partners who achieved pregnancy by natural conception (NC+, n = 10), IVF (IVF+, n = 23), or ICSI (ICSI +, n = 9) and their respective counterparts with failed pregnancy NC (n = 7), IVF (n = 23), or ICSI (n = 10), by both immunocytochemistry and flow-cytometry. Reduced expression of TRPV1 in sperm of IVF ± and ICSI ± men with respect to that NC+ men imply its role in mediating successful fertilization. Unsuccessful pregnancy outcome with an underexpression of TRPV1 in sperm of NC-/IVF-/ICSI-men suggests its role in conception and maintenance of pregnancy. Since ROS is regarded as one of the major contributors to sperm dysfunction, the effect of H2O2 +/- TRPV1 modulators (RTX/iRTX) on acrosomal reaction and calcium influx was evaluated to confirm TRPV1 as a redox sensor in human sperm. A significant increment in the percentage of acrosome reacted spermatozoa along with augmented Ca2+-influx was observed after H2O2 treatment, both in the presence or absence of TRPV1 agonist resiniferatoxin (RTX). The effect was attenuated by the TRPV1 antagonist iodoresiniferatoxin (iRTX), indicating the involvement of TRPV1 in mediating H2O2 response. Enhancement of motility and triggering of acrosomal reaction post TRPV1 activation suggested that disruption of these signaling cascades in vivo, possibly due to down-regulation of TRPV1 in these subfertile males. Bioinformatic analysis of the crosstalk between TRPV1 with fertility candidate proteins (reported to influence IVF outcome) revealed cell death and survival, cellular compromise, and embryonic development to be the primary networks affected by anomalous TRPV1 expression. We therefore postulate that TRPV1 can act as a redox sensor, and its expression in spermatozoa may serve as a fertility marker.</p