Veratridine Can Bind to a Site at the Mouth of the Channel Pore at Human Cardiac Sodium Channel NaV1.5

The cardiac sodium ion channel (NaV1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na+ ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits NaV1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on NaV1.5, we performed docking calculations and high-throughput electrophysiology experiments. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of NaV1.4 and NaV1.5 blocking drugs in experimental structures. The second site was at the “mouth” of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at NaV1.5.

Exploring Kv1.2 Channel Inactivation Through MD Simulations and Network Analysis

The KCNA2 gene encodes the Kv1.2 channel, a mammalian Shaker-like voltage-gated K+ channel, whose defections are linked to neuronal deficiency and childhood epilepsy. Despite the important role in the kinetic behavior of the channel, the inactivation remained hereby elusive. Here, we studied the Kv1.2 inactivation via a combined simulation/network theoretical approach that revealed two distinct pathways coupling the Voltage Sensor Domain and the Pore Domain to the Selectivity Filter. Additionally, we mutated some residues implicated in these paths and we explained microscopically their function in the inactivation mechanism by computing a contact map. Interestingly, some pathological residues shown to impair the inactivation lay on the paths. In summary, the presented results suggest two pathways as the possible molecular basis of the inactivation mechanism in the Kv1.2 channel. These pathways are consistent with earlier mutational studies and known mutations involved in neuronal channelopathies.

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site

Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP-N-acetylglucosamine to yield corresponding midecamycin 2′-O-glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP-N-acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2′-O-glucopyranoside, midecamycin 2′-O-GlcNAc and midecamycin 2′-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2′-O-glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.

Viral suppressors from members of the family Closteroviridae combating antiviral RNA silencing: a tale of a sophisticated arms race in host-pathogen interactions

RNA silencing is an evolutionarily homology-based gene inactivation mechanism and plays critical roles in plant immune responses to acute or chronic virus infections, which often pose serious threats to agricultural productions. Plant antiviral immunity is triggered by virus-derived small interfering RNAs (vsiRNAs) and functions to suppress virus further replication via a sequence-specific degradation manner. Through plant-virus arms races, many viruses have evolved specific protein(s), known as viral suppressors of RNA silencing (VSRs), to combat plant antiviral responses. Numerous reports have shown that VSRs can efficiently curb plant antiviral defense response via interaction with specific component(s) involved in the plant RNA silencing machinery. Members in the family Closteroviridae (closterovirids) are also known to encode VSRs to ensure their infections in plants. In this review, we will focus on the plant antiviral RNA silencing strategies, and the most recent developments on the multifunctional VSRs encoded by closterovirids. Additionally, we will highlight the molecular characters of phylogenetically-associated closterovirids, the interactions of these viruses with their host plants and transmission vectors, and epidemiology.

Photocatalytic Bactericidal Performance of LaFeO3 under Solar Light in the Presence of Natural Organic Matter: Spectroscopic and Mechanistic Evaluation

Solar photocatalytic inactivation (SPCI) of E. coli as the indicator microorganism using LaFeO3 (LF) has already been investigated under various experimental conditions, excluding any role of natural organic matter (NOM). However, comprehensive information about the behavior of E. coli and its inactivation mechanism in the presence of NOM, as well as the behavior of NOM components via solar photocatalysis using LF as a photocatalyst, has prime importance in understanding real natural water environments. Therefore, in this study, further assessment was devoted to explore the influence of various NOM representatives on the SPCI of E. coli by using LF as a novel non-TiO2 photocatalyst. The influence of NOM as well as its sub-components, such as humic acids (HA) and fulvic acids (FA), was also investigated to understand different NOM-related constituents of real natural water conditions. In addition to spectroscopic and mechanistic investigations of cell-derived organics, excitation emission matrix (EEM) fluorescence spectra with parallel factor multiway analysis (PARAFAC) modeling revealed further information about the occurrence and/or disappearance of NOM-related and bacteria-related fluorophores upon LF SPCI. Both the kinetics as well as the mechanism of the LF SPCI of E. coli in the presence of NOM compounds displayed substrate-specific variations under all conditions.

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme are involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

Evaluation of Structural Changes and Molecular Mechanism Induced by High Hydrostatic Pressure in Enterobacter sakazakii

The contamination of infant milk and powder with Enterobacter sakazakii poses a risk to human health and frequently caused recalls of affected products. This study aims to explore the inactivation mechanism of E. sakazakii induced by high hydrostatic pressure (HHP), which, unlike conventional heat treatment, is a nonthermal technique for pasteurization and sterilization of dairy food without deleterious effects. The mortality of E. sakazakii under minimum reaction conditions (50 MPa) was 1.42%, which was increased to 33.12% under significant reaction conditions (400 MPa). Scanning electron microscopy (SEM) and fluorescent staining results showed that 400 MPa led to a loss of physical integrity of cell membranes as manifested by more intracellular leakage of nucleic acid, intracellular protein and K+. Real-time quantitative PCR (RT-qPCR) analysis presents a downregulation of three functional genes (glpK, pbpC, and ompR), which were involved in cell membrane formation, indicating a lower level of glycerol utilization, outer membrane protein assembly, and environmental tolerance. In addition, the exposure of E. sakazakii to HHP modified oxidative stress, as reflected by the high activity of catalase and super oxide dismutase. The HHP treatment lowered down the gene expression of flagellar proteins (fliC, flgI, fliH, and flgK) and inhibited biofilm formation. These results determined the association of genotype to phenotype in E. sakazakii induced by HHP, which was used for the control of food-borne pathogens.

Contribution of the Adenosine 2A Receptor to Behavioral Effects of Tetrahydrocannabinol, Cannabidiol and PECS-101

The cannabis-derived molecules, ∆9 tetrahydrocannabinol (THC) and cannabidiol (CBD), are both of considerable therapeutic interest for a variety of purposes, including to reduce pain and anxiety and increase sleep. In addition to their other pharmacological targets, both THC and CBD are competitive inhibitors of the equilibrative nucleoside transporter-1 (ENT-1), a primary inactivation mechanism for adenosine, and thereby increase adenosine signaling. The goal of this study was to examine the role of adenosine A2A receptor activation in the effects of intraperitoneally administered THC alone and in combination with CBD or PECS-101, a 4′-fluorinated derivative of CBD, in the cannabinoid tetrad, elevated plus maze (EPM) and marble bury assays. Comparisons between wild-type (WT) and A2AR knock out (A2AR-KO) mice were made. The cataleptic effects of THC were diminished in A2AR-KO; no other THC behaviors were affected by A2AR deletion. CBD (5 mg/kg) potentiated the cataleptic response to THC (5 mg/kg) in WT but not A2AR-KO. Neither CBD nor THC alone affected EPM behavior; their combination produced a significant increase in open/closed arm time in WT but not A2AR-KO. Both THC and CBD reduced the number of marbles buried in A2AR-KO but not WT mice. Like CBD, PECS-101 potentiated the cataleptic response to THC in WT but not A2AR-KO mice. PECS-101 also reduced exploratory behavior in the EPM in both genotypes. These results support the hypothesis that CBD and PECS-101 can potentiate the cataleptic effects of THC in a manner consistent with increased endogenous adenosine signaling.