Structural Basis for Oxidized Glutathione Recognition by Yeast Cadmium Factor 1.

Transporters from the ABCC family have an essential role in detoxifying electrophilic compounds, including heavy metals and drugs, often after conjugation with glutathione. The Yeast Cadmium Factor 1 (Ycf1) transports glutathione conjugated to toxic heavy metals including Cd, Hg, and the metalloid As into the vacuole to safely sequester them in the cell. It also has a major role in transporting oxidized glutathione to help maintain redox balance and recycle glutathione.

Molecular mechanism of substrate transport by human peroxisomal ABCD3.

Peroxisomes are eukaryotic oxidative organelles involved in numerous metabolic functions that include fatty acid oxidation, bile acid synthesis, and detoxification of reactive oxygen species. ATP-binding cassette transporters of the D subfamily (ABCD1-3) mediate the import of CoA thioesters of fatty acids into the peroxisome. ABCD3, the most abundant of these transporters in the peroxisomal membrane, facilitates the transport of a broad spectrum of substrates including branched-chain fatty acids, very long-chain fatty acids, bile salt intermediates, and dicarboxylic acids.

Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporter efflux protein A.

A broad chemical genetic screen in Mycobacterium tuberculosis (Mtb) identified compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition, we determined the structures of EfpA with these inhibitors bound at 2.

Structural basis of aquaporin-4 autoantibody binding in neuromyelitis optica.

Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system where pathogenic autoantibodies target the water channel aquaporin-4 on human astrocytes causing neurological impairment. Autoantibody binding leads to complement-dependent and complement-independent cytotoxicity, ultimately resulting in astrocyte death, demyelination, and neuronal loss. Aquaporin-4 assembles in astrocyte plasma membranes as symmetric tetramers or as arrays of tetramers.

Structural basis of disease mutation and substrate recognition by the human SLC2A9 transporter.

Urate provides ~50% of the reducing potential in human and primate plasma which is key to detoxifying reactive oxygen by-products of cellular metabolism. Urate is the endpoint of purine metabolism in primates, and its concentration in plasma is a balance between excretion from kidney and intestine, and subsequent reabsorption in and through cells of kidney proximal tubules to maintain a regulated concentration in plasma. SLC2A9 is the primary transporter that returns urate from the basolateral side of kidney tubule cells back to plasma.

Structure and inhibition mechanisms of essential transporter efflux protein A.

A broad chemical genetics screen in to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.

Structural Basis of Aquaporin-4 Autoantibody Binding in Neuromyelitis Optica.

Neuromyelitis Optica (NMO) is an autoimmune disease of the central nervous system where pathogenic autoantibodies target the human astrocyte water channel aquaporin-4 causing neurological impairment. Autoantibody binding leads to complement dependent and complement independent cytotoxicity, ultimately resulting in astrocyte death, demyelination, and neuronal loss. Aquaporin-4 assembles in astrocyte plasma membranes as symmetric tetramers or as arrays of tetramers.

Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1.

Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism.

Structural Basis for Oxidized Glutathione Recognition by the Yeast Cadmium Factor 1.

Transporters from the ABCC family have an essential role in detoxifying electrophilic compounds including metals, drugs, and lipids, often through conjugation with glutathione complexes. The Yeast Cadmium Factor 1 (Ycf1) transports glutathione alone as well as glutathione conjugated to toxic heavy metals including Cd, Hg, and As. To understand the complicated selectivity and promiscuity of heavy metal substrate binding, we determined the cryo-EM structure of Ycf1 bound to the substrate, oxidized glutathione.

Inositol Phosphoryl Transferase, Ipt1, Is a Critical Determinant of Azole Resistance and Virulence Phenotypes in .

In this study, we have specifically blocked a key step of sphingolipid (SL) biosynthesis in by disruption of the orthologs of ScIpt1 and ScSkn1. Based on their close homology with counterparts, the proteins are predicted to catalyze the addition of a phosphorylinositol group onto mannosyl inositolphosphoryl ceramide (MIPC) to form mannosyl diinositolphosphoryl ceramide (M(IP)C), which accounts for the majority of complex SL structures in membranes. High throughput lipidome analysis confirmed the accumulation of MIPC structures in and cells, albeit to lesser extent in the latter.

Measuring Gene Expression via mRNA Abundance in Candida auris.

The recently emerged human pathogenic yeast Candida auris has become a major global threat. As compared to other Candida species, C. auris often displays a high level of resistance to commonly used antifungals and poses additional therapeutic challenges.

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation.

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood.

Unmasking of CgYor1-Dependent Azole Resistance Mediated by Target of Rapamycin (TOR) and Calcineurin Signaling in Candida glabrata.

In this study, 18 predicted membrane-localized ABC transporters of Candida glabrata were deleted individually to create a minilibrary of knockouts (KO). The transporter KOs were analyzed for their susceptibility toward antimycotic drugs. Although Cg has previously been reported to be upregulated in various azole-resistant clinical isolates of C.

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB.

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question.

A homologous overexpression system to study roles of drug transporters in Candida glabrata.

Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1, CgPDH1, CgYCF1, CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1, CgPDH1, CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus.

In vitro characterization, ADME analysis, and histological and toxicological evaluation of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains.

Background: Candida species are one of the most common causes of nosocomial bloodstream infections among the opportunistic fungi. Extensive use of antifungal agents, most of which were launched on the market more than 20 years ago, led to the selection of drug-resistant or even multidrug-resistant fungi. We recently described a novel class of antifungal macrocyclic compounds with an amidinourea moiety that is highly active against azole-resistant Candida strains.

ABC Transporter Genes Show Upregulated Expression in Drug-Resistant Clinical Isolates of : A Genome-Wide Characterization of ATP-Binding Cassette (ABC) Transporter Genes.

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including species.

Lipidomics Approaches: Applied to the Study of Pathogenesis in Candida Species.

High rate of reported cases of infections in humans caused by fungal pathogens pose serious concern. Potentially these commensal fungi remain harmless to the healthy individuals but can cause severe systemic infection in patients with compromised immune system. Effective drug remedies against these infections are rather limited.

Vacuolar Sequestration of Azoles, a Novel Strategy of Azole Antifungal Resistance Conserved across Pathogenic and Nonpathogenic Yeast.

Target alteration and overproduction and drug efflux through overexpression of multidrug transporters localized in the plasma membrane represent the conventional mechanisms of azole antifungal resistance. Here, we identify a novel conserved mechanism of azole resistance not only in the budding yeast but also in the pathogenic yeast We observed that the vacuolar-membrane-localized, multidrug resistance protein (MRP) subfamily, ATP-binding cassette (ABC) transporter of , Ybt1, could import azoles into vacuoles. Interestingly, the Ybt1 homologue in , Mlt1p, could also fulfill this function.

All about CDR transporters: Past, present, and future.

Drug resistance mechanisms in human pathogenic Candida species are continually evolving. Over the time, Candida species have acquired diverse strategies to vanquish the effects of various classes of drugs thereby, emanating as a serious life threat. Apart from the repertoire of well-established strategies, which predominantly comprise alteration, overexpression of drug targets, and chromosome duplication, Candida species have evolved a number of permeability constraints for antifungal drugs, via compromised drug import or increased drug efflux.

ABC transportome inventory of human pathogenic yeast Candida glabrata: Phylogenetic and expression analysis.

ATP-binding cassette (ABC) is one of the two major superfamilies of transporters present across the evolutionary scale. ABC superfamily members came to prominence due to their ability to extrude broad spectrum of substrates and to confer multi drug resistance (MDR). Overexpression of some ABC transporters in clinical isolates of Candida species was attributed to the development of MDR phenotypes.

Inventory of ABC proteins and their putative role in salt and drug tolerance in Debaryomyces hansenii.

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance.

Phosphatidylserine decarboxylase governs plasma membrane fluidity and impacts drug susceptibilities of Candida albicans cells.

Plasma membrane (PM) lipid composition imbalances affect drug susceptibilities of the human pathogen Candida albicans. The PM fundamental structure is made up of phospholipid bilayer where phosphatidylethanolamine (PE) contributes as second major phospholipid moieties, which is asymmetrically distributed between the two leaflets of the bilayer. PSD1 and PSD2 genes encode phosphatidylserine decarboxylase which converts phosphatidylserine (PS) into PE in C.

Azole resistance in a mutant lacking the ABC transporter depends on TOR signaling.

ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of an ABC transporter in , orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain.

pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport.

Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans This antiporter belongs to the drug/H(+) antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H(+) towards cytosol are inextricably linked processes. For monitoring the drug/H(+) antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH.