Structural basis of G protein-Coupled receptor CMKLR1 activation and signaling induced by a chemerin-derived agonist.

Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) or chemerin receptor 1, is a chemoattractant G protein-coupled receptor (GPCR) that responds to the adipokine chemerin and is highly expressed in innate immune cells, including macrophages and neutrophils. The signaling pathways of CMKLR1 can lead to both pro- and anti-inflammatory effects depending on the ligands and physiological contexts. To understand the molecular mechanisms of CMKLR1 signaling, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of the CMKLR1-Gi signaling complex with chemerin9, a nanopeptide agonist derived from chemerin, which induced complex phenotypic changes of macrophages in our assays. The cryo-EM structure, together with molecular dynamics simulations and mutagenesis studies, revealed the molecular basis of CMKLR1 signaling by elucidating the interactions at the ligand-binding pocket and the agonist-induced conformational changes. Our results are expected to facilitate the development of small molecule CMKLR1 agonists that mimic the action of chemerin9 to promote the resolution of inflammation.

The Alkylamine Stimulant 1,3-Dimethylamylamine Exhibits Substrate-Like Regulation of Dopamine Transporter Function and Localization.

The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used nonmedically as an appetite suppressant and exercise performance enhancer despite adverse cardiovascular effects that have limited its legal status. There is scant research describing the mechanism of action of DMAA, making it difficult to gauge risks or therapeutic potential. An important molecular target of structurally related phenethylamines, such as amphetamine, for regulating mood, cognition, movement, and the development of substance use disorder is the dopamine transporter, which limits the range and magnitude of dopamine signaling via reuptake from the extracellular space. The present studies were therefore initiated to characterize the effects of DMAA on dopamine transporter function. Specifically, we tested the hypothesis that DMAA exhibits substrate-like effects on dopamine transporter function and trafficking. In transport assays in human embryonic kidney cells, DMAA inhibited dopamine uptake by the human dopamine transporter in a competitive manner. Docking analysis and molecular dynamics simulations supported these findings, revealing that DMAA binds to the S1 substrate binding site and induces a conformational change from outward-facing open to outward-facing closed states, similar to the known substrates. Further supporting substrate-like effects of DMAA, the drug stimulated dopamine transporter endocytosis in a heterologous expression system via cocaine- and protein kinase A-sensitive mechanisms, mirroring findings with amphetamine. Together, these data indicate that DMAA elicits neurologic effects by binding to and regulating function of the dopamine transporter. Furthermore, pharmacologic distinctions from amphetamine reveal structural determinants for regulating transporter conformation and add mechanistic insight for the regulation of dopamine transporter endocytosis. SIGNIFICANCE STATEMENT: The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used as an appetite suppressant and athletic performance enhancer and is structurally similar to amphetamine, but there is scant research describing its mechanism of action. Characterizing the effects of DMAA on dopamine transporter function supports evaluation of potential risks and therapeutic potential while also revealing mechanistic details of dynamic transporter-substrate interactions.

Superantigenic character of an insert unique to SARS-CoV-2 spike supported by skewed TCR repertoire in patients with hyperinflammation.

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches.

Allosteric Modulator KM822 Attenuates Behavioral Actions of Amphetamine in Caenorhabditis elegans through Interactions with the Dopamine Transporter DAT-1.

Aberrant dopamine (DA) signaling is associated with several psychiatric disorders, such as autism, bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. In addition, psychostimulants, such as cocaine and D-amphetamine (AMPH), rely on the competitive interactions with the transporter's substrate binding site to produce their rewarding effects. Agents that exhibit noncompetitive, allosteric modulation of DAT remain an important topic of investigation due to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors; however, whether DAT is the sole mediator of KM822 actions in vivo is unproven given the large number of potential off-target sites. Here, we provide in silico and in vitro evidence that the allosteric site engaged by KM822 is conserved between human DAT and Caenorhabditis elegans DAT-1. KM822 binds to a similar pocket in DAT-1 as previously identified in human DAT. In functional dopamine uptake assays, KM822 affects the interaction between AMPH and DAT-1 by reducing the affinity of AMPH for DAT-1. Finally, through a combination of genetic and pharmacological in vivo approaches we provide evidence that KM822 diminishes the behavioral actions of AMPH on swimming-induced paralysis through a direct allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that Caenorhabditis elegans can be operationalized to identify and investigate the interactions of DAT allosteric modulators. SIGNIFICANCE STATEMENT: We previously demonstrated that the dopamine transporter (DAT) allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using in silico and in vivo genetic approaches, we extend this finding to interactions with amphetamine, demonstrating evolutionary conservation of the DAT allosteric site. In Caenorhabditis elegans, we report that KM822 suppresses amphetamine behavioral effects via specific interactions with DAT-1. Our findings reveal Caenorhabditis elegans as a new tool to study allosteric modulation of DAT and its behavioral consequences.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity.

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule-induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.

A systems-level study reveals host-targeted repurposable drugs against SARS-CoV-2 infection.

Understanding the mechanism of SARS-CoV-2 infection and identifying potential therapeutics are global imperatives. Using a quantitative systems pharmacology approach, we identified a set of repurposable and investigational drugs as potential therapeutics against COVID-19. These were deduced from the gene expression signature of SARS-CoV-2-infected A549 cells screened against Connectivity Map and prioritized by network proximity analysis with respect to disease modules in the viral-host interactome. We also identified immuno-modulating compounds aiming at suppressing hyperinflammatory responses in severe COVID-19 patients, based on the transcriptome of ACE2-overexpressing A549 cells. Experiments with Vero-E6 cells infected by SARS-CoV-2, as well as independent syncytia formation assays for probing ACE2/SARS-CoV-2 spike protein-mediated cell fusion using HEK293T and Calu-3 cells, showed that several predicted compounds had inhibitory activities. Among them, salmeterol, rottlerin, and mTOR inhibitors exhibited antiviral activities in Vero-E6 cells; imipramine, linsitinib, hexylresorcinol, ezetimibe, and brompheniramine impaired viral entry. These novel findings provide new paths for broadening the repertoire of compounds pursued as therapeutics against COVID-19.

Bicarbonate permeation through anion channels: its role in health and disease.

Many anion channels, frequently referred as Cl- channels, are permeable to different anions in addition to Cl-. As the second-most abundant anion in the human body, HCO3- permeation via anion channels has many important physiological roles. In addition to its classical role as an intracellular pH regulator, HCO3- also controls the activity and stability of dissolved proteins in bodily fluids such as saliva, pancreatic juice, intestinal fluid, and airway surface liquid. Moreover, HCO3- permeation through these channels affects membrane potentials that are the driving forces for transmembrane transport of solutes and water in epithelia and affect neuronal excitability in nervous tissue. Consequently, aberrant HCO3- transport via anion channels causes a number of human diseases in respiratory, gastrointestinal, genitourinary, and neuronal systems. Notably, recent studies have shown that the HCO3- permeabilities of several anion channels are not fixed and can be altered by cellular stimuli, findings which may have both physiological and pathophysiological significance. In this review, we summarize recent progress in understanding the molecular mechanisms and the physiological roles of HCO3- permeation through anion channels. We hope that the present discussions can stimulate further research into this very important topic, which will provide the basis for human disorders associated with aberrant HCO3- transport.

Allosteric modulation of human dopamine transporter activity under conditions promoting its dimerization.

The human dopamine (DA) transporter (hDAT) is a key regulator of neurotransmission and a target for antidepressants and addictive drugs. Despite the recent resolution of dDAT structures from Drosophila melanogaster, complete understanding of its mechanism of function and even information on its biological assembly is lacking. The resolved dDAT structures are monomeric, but growing evidence suggests that hDAT might function as a multimer, and its oligomerization may be relevant to addictive drug effects. Here, using structure-based computations, we examined the possible mechanisms of hDAT dimerization and its dynamics in a lipid bilayer. Using a combination of site-directed mutagenesis, DA-uptake, and cross-linking experiments that exploited the capacity of Cys-306 to form intermonomeric disulfide bridges in the presence of an oxidizing agent, we tested the effects of mutations at transmembrane segment (TM) 6 and 12 helices in HEK293 cells. The most probable structural model for hDAT dimer suggested by computations and experiments differed from the dimeric structure resolved for the bacterial homolog, LeuT, presumably because of a kink at TM12 preventing favorable monomer packing. Instead, TM2, TM6, and TM11 line the dimer interface. Molecular dynamics simulations of the dimeric hDAT indicated that the two subunits tend to undergo cooperative structural changes, both on local (extracellular gate opening/closure) and global (transition between outward-facing and inward-facing states) scales. These observations suggest that hDAT transport properties may be allosterically modulated under conditions promoting dimerization. Our study provides critical insights into approaches for examining the oligomerization of neurotransmitter transporters and sheds light on their drug modulation.

Pore dilatation increases the bicarbonate permeability of CFTR, ANO1 and glycine receptor anion channels.

KEY POINTS: Cellular stimuli can modulate the ion selectivity of some anion channels, such as CFTR, ANO1 and the glycine receptor (GlyR), by changing pore size. Ion selectivity of CFTR, ANO1 and GlyR is critically affected by the electric permittivity and diameter of the channel pore. Pore size change affects the energy barriers of ion dehydration as well as that of size-exclusion of anion permeation. Pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of CFTR, ANO1 and GlyR. Dynamic change in P HC O3/ Cl may mediate many physiological and pathological processes. ABSTRACT: Chloride (Cl(-) ) and bicarbonate (HCO3 (-) ) are two major anions and their permeation through anion channels plays essential roles in our body. However, the mechanism of ion selection by the anion channels is largely unknown. Here, we provide evidence that pore dilatation increases the bicarbonate permeability (P HC O3/ Cl ) of anion channels by reducing energy barriers of size-exclusion and ion dehydration of HCO3 (-) permeation. Molecular, physiological and computational analyses of major anion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR), anoctamin-1(ANO1/TMEM16A) and the glycine receptor (GlyR), revealed that the ion selectivity of anion channels is basically determined by the electric permittivity and diameter of the pore. Importantly, cellular stimuli dynamically modulate the anion selectivity of CFTR and ANO1 by changing the pore size. In addition, pore dilatation by a mutation in the pore-lining region alters the anion selectivity of GlyR. Changes in pore size affected not only the energy barriers of size exclusion but that of ion dehydration by altering the electric permittivity of water-filled cavity in the pore. The dynamic increase in P HC O3/ Cl by pore dilatation may have many physiological and pathophysiological implications ranging from epithelial HCO3 (-) secretion to neuronal excitation.

Structure-Encoded Global Motions and Their Role in Mediating Protein-Substrate Interactions.

Recent structure-based computational studies suggest that, in contrast to the classical description of equilibrium fluctuations as wigglings and jigglings, proteins have access to well-defined spectra of collective motions, called intrinsic dynamics, encoded by their structure under native state conditions. In particular, the global modes of motions (at the low frequency end of the spectrum) are shown by multiple studies to be highly robust to minor differences in the structure or to detailed interactions at the atomic level. These modes, encoded by the overall fold, usually define the mechanisms of interactions with substrates. They can be estimated by low-resolution models such as the elastic network models (ENMs) exclusively based on interresidue contact topology. The ability of ENMs to efficiently assess the global motions intrinsically favored by the overall fold as well as the relevance of these predictions to the dominant changes in structure experimentally observed for a given protein in the presence of different substrates suggest that the intrinsic dynamics plays a role in mediating protein-substrate interactions. These observations underscore the functional significance of structure-encoded dynamics, or the importance of the predisposition to favor functional global modes in the evolutionary selection of structures.

Complete mapping of substrate translocation highlights the role of LeuT N-terminal segment in regulating transport cycle.

Neurotransmitter: sodium symporters (NSSs) regulate neuronal signal transmission by clearing excess neurotransmitters from the synapse, assisted by the co-transport of sodium ions. Extensive structural data have been collected in recent years for several members of the NSS family, which opened the way to structure-based studies for a mechanistic understanding of substrate transport. Leucine transporter (LeuT), a bacterial orthologue, has been broadly adopted as a prototype in these studies. This goal has been elusive, however, due to the complex interplay of global and local events as well as missing structural data on LeuT N-terminal segment. We provide here for the first time a comprehensive description of the molecular events leading to substrate/Na+ release to the postsynaptic cell, including the structure and dynamics of the N-terminal segment using a combination of molecular simulations. Substrate and Na+-release follows an influx of water molecules into the substrate/Na+-binding pocket accompanied by concerted rearrangements of transmembrane helices. A redistribution of salt bridges and cation-π interactions at the N-terminal segment prompts substrate release. Significantly, substrate release is followed by the closure of the intracellular gate and a global reconfiguration back to outward-facing state to resume the transport cycle. Two minimally hydrated intermediates, not structurally resolved to date, are identified: one, substrate-bound, stabilized during the passage from outward- to inward-facing state (holo-occluded), and another, substrate-free, along the reverse transition (apo-occluded).

Exploring the conformational transitions of biomolecular systems using a simple two-state anisotropic network model.

Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results.

Energy landscape of LeuT from molecular simulations.

The bacterial sodium-coupled leucine transporter (LeuT) has been broadly used as a structural model for understanding the structure-dynamics-function of mammalian neurotransmitter transporters as well as other solute carriers that share the same fold (LeuT fold), as the first member of the family crystallographically resolved in multiple states: outward-facing open, outward-facing occluded, and inward-facing open. Yet, a complete picture of the energy landscape of (sub)states visited along the LeuT transport cycle has been elusive. In an attempt to visualize the conformational spectrum of LeuT, we performed extensive simulations of LeuT dimer dynamics in the presence of substrate (Ala or Leu) and co-transported Na(+) ions, in explicit membrane and water. We used both conventional molecular dynamics (MD) simulations (with Anton supercomputing machine) and a recently introduced method, collective MD, that takes advantage of collective modes of motions predicted by the anisotropic network model. Free energy landscapes constructed based on ∼40 µs trajectories reveal multiple substates occluded to the extracellular (EC) and/or intracellular (IC) media, varying in the levels of exposure of LeuT to EC or IC vestibules. The IC-facing transmembrane (TM) helical segment TM1a shows an opening, albeit to a smaller extent and in a slightly different direction than that observed in the inward-facing open crystal structure. The study provides insights into the spectrum of conformational substates and paths accessible to LeuT and highlights the differences between Ala- and Leu-bound substates.

Structural mechanisms for VMAT2 inhibition by tetrabenazine.

The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson's disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2's importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here we report a 3.1 Å resolution cryo-EM structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntington's chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.

Structural mechanisms for VMAT2 inhibition by tetrabenazine.

The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson's disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2's importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here, we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntington's chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.

Allosteric modulation of serotonin and dopamine transporters: New insights from computations and experiments.

Human monoamine transporters (MATs) are critical to regulating monoaminergic neurotransmission by translocating their substrates from the synaptic space back into the presynaptic neurons. As such, their primary substrate binding site S1 has been targeted by a wide range of compounds for treating neuropsychiatric and neurodegenerative disorders including depression, ADHD, neuropathic pain, and anxiety disorders. We present here a comparative study of the structural dynamics and ligand-binding properties of two MATs, dopamine transporter (DAT) and serotonin transporter (SERT), with focus on the allosteric modulation of their transport function by drugs or substrates that consistently bind a secondary site S2, proposed to serve as an allosteric site. Our systematic analysis of the conformational space and dynamics of a dataset of 50 structures resolved for DAT and SERT in the presence of one or more ligands/drugs reveals the specific residues playing a consistent role in coordinating the small molecules bound to subsites S2-I and S2-II within S2, such as R476 and Y481 in dDAT and E494, P561, and F556 in hSERT. Further analysis reveals how DAT and SERT differ in their two principal modes of structural changes, PC1 and PC2. Notably, PC1 underlies the transition between outward- and inward-facing states of the transporters as well as their gating; whereas PC2 supports the rearrangements of TM helices near the S2 site. Finally, the examination of cross-correlations between structural elements lining the respective sites S1 and S2 point to the crucial role of coupled motions between TM6a and TM10. In particular, we note the involvement of hSERT residues F335 and G338, and E493-E494-T497 belonging to these two respective helices, in establishing the allosteric communication between S1 and S2. These results help understand the molecular basis of the action of drugs that bind to the S2 site of DAT or SERT. They also provide a basis for designing allosteric modulators that may provide better control of specific interactions and cellular pathways, rather than indiscriminately inhibiting the transporter by targeting its orthosteric site.

Synthesis and Structure-Activity Relationships for Glutamate Transporter Allosteric Modulators.

Excitatory amino acid transporters (EAATs) are essential CNS proteins that regulate glutamate levels. Excess glutamate release and alteration in EAAT expression are associated with several CNS disorders. Previously, we identified positive allosteric modulators (PAM) of EAAT2, the main CNS transporter, and have demonstrated their neuroprotective properties in vitro. Herein, we report on the structure-activity relationships (SAR) for the analogs identified from virtual screening and from our medicinal chemistry campaign. This work identified several selective EAAT2 positive allosteric modulators (PAMs) such as compounds 4 (DA-023) and 40 (NA-014) from a library of analogs inspired by GT949, an early generation compound. This series also provides nonselective EAAT PAMs, EAAT inhibitors, and inactive compounds that may be useful for elucidating the mechanism of EAAT allosteric modulation.

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension.

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease.

Coupled global and local changes direct substrate translocation by neurotransmitter-sodium symporter ortholog LeuT.

Significant advances have been made in recent years in characterizing neurotransmitter:sodium symporter (NSS) family structure and function. Yet, many time-resolved events and intermediates that control the various stages of transport cycle remain to be elucidated. Whether NSSs harbor one or two sites for binding their substrates (neurotransmitters or amino acids), and what the role of the secondary site S2 is, if any, are still unresolved. Using molecular modeling and simulations for LeuT, a bacterial NSS, we present a comprehensive account of substrate-binding and -stabilization events, and subsequently triggered interactions leading to substrate (alanine) release. LeuT instantaneous conformation as it reconfigures from substrate-receiving (outward-facing) to -releasing (inward-facing) state appears to be a determinant of its affinity to bind substrate at site S2. In the outward-facing state, S1 robustly binds alanine and regulates subsequent redistribution of interactions to trigger extracellular gate closure; whereas S2 is only a transient binding site. The substrate-binding affinity at S2 increases in an intermediate close to inward-facing state. LeuT harbors the two substrate-binding sites, and small displacements of second substrate near S2 are observed to induce concerted small translocations in the substrate bound to primary site S1, although complete release requires collective structural rearrangements that fully expose the intracellular vestibule to the cytoplasm.