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While drugs and other industrial chemicals are routinely studied to assess risks, many widely used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cellular function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM, transcriptomics and, mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, which is involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to MCHM, and loss of Yke4 decreased MCHM tolerance. The addition of zinc to MCHM-sensitive yeast rescued growth inhibition. In vitro assays demonstrated that MCHM acted as a hydrotrope and prevented protein interactions, while zinc induced the aggregation of proteins. We hypothesized that MCHM altered the structures of extracellular domains of proteins, and the addition of zinc stabilized the structure to maintain metal homeostasis in yeast exposed to MCHM. Electronic supplementary material The online version of this article (10.1007/s12011-019-01850-z) contains supplementary material, which is available to authorized users.

The main role of voltage-gated proton channels (Hv1) is to extrude protons from the intracellular milieu when, mediated by different cellular processes, the H+ concentration increases. Hv1 are exquisitely selective for protons and their structure is homologous to the voltage sensing domain (VSD) of other voltage-gated ion channels like sodium, potassium, and calcium channels. In clear contrast to the classical voltage-dependent channels, Hv1 lacks a pore domain and thus permeation necessarily occurs through the voltage sensing domain. Hv1 channels are activated by depolarizing voltages, and increases in internal proton concentration. It has been proposed that local conformational changes of the transmembrane segment S4, driven by depolarization, trigger the molecular rearrangements that open Hv1. However, it is still unclear how the electromechanical coupling is achieved between the VSD and the potential pore, allowing the proton flux from the intracellular to the extracellular side. Here we provide a revised view of voltage activation in Hv1 channels, offering a comparative scenario with other voltage sensing channels domains.

Carbon monoxide (CO) is a gaseous transmitter that is known to be involved in several physiological processes, but surprisingly it is also becoming a promising molecule to treat several pathologies including stroke and cancer. CO can cross the plasma membrane and activate guanylate cyclase, increasing the cGMP concentration and activating some kinases, including PKG. The other mechanism of action involves induction of protein carbonylation. CO is known to directly and indirectly modulate the function of ion channels at the plasma membrane, which in turn have important repercussions in the cellular behavior. One group of these channels is hemichannels, which are formed by proteins known as connexins (Cxs). Hemichannel allows not only the flow of ions through their pore but also the release of molecules such as ATP and glutamate. Therefore, their modulation not only impacts cellular function but also cellular communication, having the capability to affect tissular behavior. Here, we review the most recent results regarding the effect of CO on Cx hemichannels and their possible repercussions on pathologies. © 2015 IUBMB Life, 67(6):428–437, 2015

Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and propylene glycol phenol ether (PPH) were accidentally released into the Elk River, West Virginia, contaminating the tap water of around 300,000 residents. Crude MCHM is an industrial chemical used as flotation reagent to clean coal. At the time of the spill, MCHM’s toxicological data were limited, an issue that have been addressed by different studies focused on understanding the immediate and long-term effects of MCHM on human health and the environment. UsingS. cerevisiaeas a model organism we study the effect of acute exposition to crude MCHM on metabolism. Yeasts were treated with MCHM 3.9 mM in YPD for 30 minutes. Polar and lipid metabolites were extracted from cells by a chloroform-methanol-water mixture. The extracts were then analyzed by direct injection ESI-MS and by GC-MS. The metabolomics analysis was complemented with flux balance analysis simulations done with genome-scale metabolic network models (GSMNM) of MCHM treated vs non-treated control. We integrated the effect of MCHM on yeast gene expression from RNA-Seq data within these GSMNM. 181 and 66 metabolites were identified by the ESI-MS and GC-MS procedures, respectively. From these 38 and 34 relevant metabolites were selected from ESI-MS and GC-MS respectively, for 72 unique compounds. MCHM induced amino acid accumulation, via its effects on amino acid metabolism, as well as a potential impairment of ribosome biogenesis. MCHM affects phospholipid biosynthesis and decrease the levels of ergosterol, with a potential impact in the biophysical properties of yeast cellular membranes. The FBA simulations were able to reproduce the deleterious effect of MCHM on cell’s growth and suggest that the effect of MCHM on ubiquinol:ferricytochrome c reductase reaction, caused by the under-expression ofCYT1gene, could be the driven force behind the observed effect on yeast metabolism and growth.

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity.

Plasmepsins are aspartic proteases involved in the initial steps of the hemoglobin degradation pathway, a critical stage in the Plasmodium falciparum life cycle during human infection. Thus, they are attractive targets for novel therapeutic compounds to treat malaria, which remains one of the world's biggest health problems. The three-dimensional structures available for P. falciparum plasmepsins II and IV make structure-based drug design of antimalarial compounds that focus on inhibiting plasmepsins possible. However, the structural flexibility of the plasmepsin active site cavity combined with insufficient knowledge of the functional residues and of those determining the specificity of parasitic enzymes is a drawback when designing specific inhibitors. In this study, we have combined a sequence and structural analysis with molecular dynamics simulations to predict the functional residues in P. falciparum plasmepsins. The careful analysis of X-ray structures and 3D models carried out here suggests that residues Y17, V105, T108, L191, L242, Q275, and T298 are important for plasmepsin function. These seven amino acids are conserved across the malarial strains but not in human aspartic proteases. Residues V105 and T108 are localized in a flap of an interior pocket and they only establish contacts with a specific non-peptide achiral inhibitor. We also observed a rapid conformational change in the L3 region of plasmepsins that closes the active site of the enzyme, which explains earlier experimental findings. These results shed light on the role of V105 and T108 residues in plasmepsin specificities, and they should be useful in structure-based design of novel, selective inhibitors that may serve as antimalarial drugs. Proteins 2008. © 2008 Wiley-Liss, Inc.

The keratitis-ichthyosis-deafness (KID) syndrome is characterized by corneal, skin, and hearing abnormalities. KID has been linked to heterozygous dominant missense mutations in the GJB2 and GJB6 genes, encoding connexin26 and 30, respectively. In vitro evidence indicates that KID mutations lead to hyperactive (open) hemichannels, which in some cases is accompanied by abnormal function of gap junction channels. Transgenic mouse models expressing connexin26 KID mutations reproduce human phenotypes and present impaired epidermal calcium homeostasis and abnormal lipid composition of the stratum corneum affecting the water barrier. Here we have compiled relevant data regarding the KID syndrome and propose a mechanism for the epidermal aspects of the disease.

Sticholysins I and II (Sts I and II) are two potent cytolysins from the sea anemone Stichodactyla helianthus. These isoforms present 13 substitutions, with three non-conservative located at the N-terminus. St II is considerably more hemolytic than St I in human red blood cells, a result explained by the smaller number of negatively charged groups present at St II's N-terminus. In the present work, we have obtained a recombinant St I (rSt I), differing from the wild type in a single amino acid residue (E16Q). This pseudo-wild type is structurally similar to St I and shows a similar capacity to interact with and form pores in model membranes. This was assessed by the intrinsic fluorescence increase in the presence of liposomes, their adsorption to bilayers (measured by SPR), their concentration at the air-water interface, their interaction with lipid monolayers and their capacity to promote the release of carboxyfluorescein entrapped in liposomes. In spite of these similarities, rSt I presents a larger hemolytic activity in human red blood cells than St I, being intermediate in activity between Sts I and II. The results obtained in the present work emphasize that even the change of one single E by Q at the N-terminal segment may modify the toxin HA and show that this functional property is the most sensitive to subtle changes in the protein primary structure.

Voltage-gated proton channels are integral membrane proteins with the capacity to permeate elementary particles in a voltage and pH dependent manner. These proteins have been found in several species and are involved in various physiological processes. Although their primary topology is known, lack of details regarding their structures in the open conformation has limited analyses toward a deeper understanding of the molecular determinants of their function and regulation. Consequently, the function-structure relationships have been inferred based on homology models. In the present work, we review the existing proton channel models, their assumptions, predictions and the experimental facts that support them. Modeling proton channels is not a trivial task due to the lack of a close homolog template. Hence, there are important differences between published models. This work attempts to critically review existing proton channel models toward the aim of contributing to a better understanding of the structural features of these proteins.

Parallel ligand- and structure-based virtual screenings of 269 steroids with anabolic activity evaluated in vivo were performed. The quantitative structure-activity relationship (QSAR) model expressed by selected descriptors as the octanol-water partition coefficient, the molar volume and the quantum mechanical calculated charge values on atoms C1, C2, C5, C9, C10, C14 and C17 of the steroid skeleton, expresses structural features of anabolic steroids (AS) contributing to the transport and steroid-receptor interaction. On the other hand, computational simulations of a candidate ligand binding to a receptor study (a "docking" procedure) predict the association of these AS with the human androgen receptor (AR). Fourteen compounds were identified as lead; the most potent was the 7α-methylestr-4-en-3, 17-dione. It was concluded that a good anabolic activity requires hydrogen bonding interactions between both Arg752 and Gln711 residues in the cycles A with O3 atom of the steroid and either Asn705 and Thr877 residues in the cycles D of steroid with O17 atom.

The structurally related gangliosides N-glycolyl GM3 and N-acetyl GM3 are potential targets for tumor immunotherapy. 14F7 is a monoclonal antibody able to discriminate the tumor-specific antigen N-glycolyl GM3 from the closely related N-acetyl GM3 on the basis of the presence of a single additional hydroxyl group in the former. A combinatorial phage display strategy, based on the screening of a large library followed by refined mutagenesis, allowed a thorough exploration of the binding chemistry of this unique antibody. Three essential features of the heavy chain variable region were identified: two aromatic rings (in positions 33 and 100D) contributing to the binding site architecture and an arginine residue (position 98) critical for recognition. Directed evolution of 14F7 resulted in novel variants that cross-react with the tumor-associated antigen N-acetyl GM3 and display recurrent replacements: the substitution W33Q and the appearance of additional arginine residues at several positions of CDR H1. Successful conversion of such engineered variable regions into whole cross-reactive anti-GM3 immunoglobulins validated our phage-based approach to study and modify the lead antibody 14F7. The resulting family of closely related antibodies offers new tools to study the mechanisms of cell death induced by antibodies targeting gangliosides. In vitro directed evolution was useful to overcome the technical limitations to obtain anti-ganglioside antibodies. The case of 14F7 illustrates the power of combining library screening with focused site-directed randomization for a comprehensive scanning of protein interactions.

Of the ∼22,000 protein-coding genes in the human genome, an estimated 8,000 are druggable (1). The current drug market targets no more than 450 unique human proteins (1) and many contain antihypertensive, antineoplastic, or anti-inflammatory effects. Receptors, enzymes, and transporter proteins comprise most of the targets. The following protein families are overrepresented: rhodopsin-like G protein-coupled receptors, voltage-gated ion channels, and ligand-gated ion channels (1). Their membrane localization, diverse tissue distribution, and the critical roles played by ion channels in human physiology makes these proteins attractive targets for drug discovery (2). Although worldwide sales of ion channel-targeted drugs are over US$10 billion, ion channels remain significantly underexploited as therapeutic targets (3).

Phage-displayed peptides recognized by two monoclonal antibodies against glucitollysine were selected. The most prominent feature of the peptide panel was the presence of paired Cys in most of them (21/24 peptides). The availability of a wide variety of peptides having differently spaced paired Cys, as well as truly linear Cys-free peptides, gave the opportunity to explore the role of disulfide bridges in phage selection. Some Cys-containing peptides came from a Cys-flanked cyclic 9-mer library, but most of them (18/21) were derived from a totally random 12-mer library, and hence the presence of Cys was dictated by the selector antibodies. Motifs shared by several peptides (potentially involved in binding) often contained or were flanked by Cys residues. Binding of all Cys-containing phage-displayed peptides was abolished/decreased after a reducing treatment. Screening a random peptide library (without invariant Cys residues) is powerful enough to clearly reveal the need, preferences, and diversity of Cys-mediated structural constraints for recognition.