Insights into autoregulation of a membrane protein complex by its cytoplasmic domains.

Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques, especially if they lack a single, defined structure. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used microsecond-scale molecular simulations of the BetP trimer to compare reported conformations of the 45-amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from electron microscopy (EM) data represents a more stable global orientation of the C-terminal segment under downregulating conditions while also providing a detailed molecular description of its dynamics and highlighting specific interactions with lipids, ions, and neighboring transporter subunits. A missing piece of the molecular puzzle is the N-terminal segment, whose dynamic nature has prevented structural characterization. Using Rosetta to generate ensembles of de novo conformations in the context of the EM-derived structure robustly identifies two features of the N-terminal tail, namely 1) short helical elements and 2) an orientation that would confine potential interactions to the protomer in the counterclockwise direction (viewed from the cytoplasm). Since each C-terminal tail only contacts the protomer in the clockwise direction, these results indicate an intricate interplay between the three protomers of BetP in the downregulated protein and a multidirectionality that may facilitate autoregulation of transport.

Disrupted Choline Clearance and Sustained Acetylcholine Release In Vivo by a Common Choline Transporter Coding Variant Associated with Poor Attentional Control in Humans.

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional control as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, in vivo, CHT-mediated choline transport, and ACh release. Relative to wild-type (WT) mice, CHT-mediated clearance of choline in male and female mice expressing one or two Val89 alleles was reduced by over 80% in cortex and over 50% in striatum. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. When challenged with a visual disruptor to reveal attentional control mechanisms, Val89 mice failed to adopt a more conservative response bias. Structural modeling revealed that Val89 may attenuate choline transport by altering conformational changes of CHT that support normal transport rates. Our findings support the view that diminished sustained cholinergic signaling capacity underlies perturbed attentional performance in individuals expressing CHT Val89. The CHT Val89 mouse serves as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.SIGNIFICANCE STATEMENT Acetylcholine (ACh) signaling depends on the functional capacity of the neuronal choline transporter (CHT). Previous research demonstrated that humans expressing the common CHT coding variant Val89 exhibit attentional vulnerabilities and attenuated fronto-cortical activation during attention. Here, we find that mice engineered to express the Val89 variant exhibit reduced CHT-mediated choline clearance and a diminished capacity to sustain ACh release. Additionally, Val89 mice lack cognitive flexibility in response to an attentional challenge. These findings provide a mechanistic and cognitive framework for interpreting the attentional phenotype associated with the human Val89 variant and establish a model that permits a more invasive interrogation of CNS effects as well as the development of therapeutic strategies for those, including Val89 carriers, with presynaptic cholinergic perturbations.

CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct.

Enlargement of the endolymphatic sac, duct, and vestibular aqueduct (EVA) is the most common inner ear malformation identified in patients with sensorineural hearing loss. EVA is associated with pathogenic variants in SLC26A4. However, in European-Caucasian populations, about 50% of patients with EVA carry no pathogenic alleles of SLC26A4. We tested for the presence of variants in CHD7, a gene known to be associated with CHARGE syndrome, Kallmann syndrome, and hypogonadotropic hypogonadism, in a cohort of 34 families with EVA subjects without pathogenic alleles of SLC26A4. In two families, NM_017780.4: c.3553A > G [p.(Met1185Val)] and c.5390G > C [p.(Gly1797Ala)] were detected as monoallelic CHD7 variants in patients with EVA. At least one subject from each family had additional signs or potential signs of CHARGE syndrome but did not meet diagnostic criteria for CHARGE. In silico modeling of these two missense substitutions predicted detrimental effects upon CHD7 protein structure. Consistent with a role of CHD7 in this tissue, Chd7 transcript and protein were detected in all epithelial cells of the endolymphatic duct and sac of the developing mouse inner ear. These results suggest that some CHD7 variants can cause nonsyndromic hearing loss and EVA. CHD7 should be included in DNA sequence analyses to detect pathogenic variants in EVA patients. Chd7 expression and mutant phenotype data in mice suggest that CHD7 contributes to the formation or function of the endolymphatic sac and duct.

New insights into Perrault syndrome, a clinically and genetically heterogeneous disorder.

Hearing loss and impaired fertility are common human disorders each with multiple genetic causes. Sometimes deafness and impaired fertility, which are the hallmarks of Perrault syndrome, co-occur in a person. Perrault syndrome is inherited as an autosomal recessive disorder characterized by bilateral mild to severe childhood sensorineural hearing loss with variable age of onset in both sexes and ovarian dysfunction in females who have a 46, XX karyotype. Since the initial clinical description of Perrault syndrome 70 years ago, the phenotype of some subjects may additionally involve developmental delay, intellectual deficit and other neurological disabilities, which can vary in severity in part dependent upon the genetic variants and the gene involved. Here, we review the molecular genetics and clinical phenotype of Perrault syndrome and focus on supporting evidence for the eight genes (CLPP, ERAL1, GGPS1, HARS2, HSD17B4, LARS2, RMND1, TWNK) associated with Perrault syndrome. Variants of these eight genes only account for approximately half of the individuals with clinical features of Perrault syndrome where the molecular genetic base remains under investigation. Additional environmental etiologies and novel Perrault disease-associated genes remain to be identified to account for unresolved cases. We also report a new genetic variant of CLPP, computational structural insight about CLPP and single cell RNAseq data for eight reported Perrault syndrome genes suggesting a common cellular pathophysiology for this disorder. Some unanswered questions are raised to kindle future research about Perrault syndrome.

Amphetamines signal through intracellular TAAR1 receptors coupled to Gα13 and GαS in discrete subcellular domains.

The extensive use of amphetamines to treat attention deficit hyperactivity disorders in children provides a compelling rationale for understanding the mechanisms of action of amphetamines and amphetamine-related drugs. We have previously shown that acute amphetamine (AMPH) regulates the trafficking of both dopamine and glutamate transporters in dopamine neurons by increasing activation of the small GTPase RhoA and of protein kinase A. Here we demonstrate that these downstream signaling events depend upon the direct activation of a trace amine-associated receptor, TAAR1, an intracellular G-protein coupled receptor (GPCR) that can be activated by amphetamines, trace amines, and biogenic amine metabolites. Using cell lines and mouse lines in which TAAR1 expression has been disrupted, we demonstrate that TAAR1 mediates the effects of AMPH on both RhoA and cAMP signaling. Inhibition of different Gα signaling pathways in cell lines and in vivo using small cell-permeable peptides confirms that the endogenous intracellular TAAR1 couples to G13 and to GS α-subunits to increase RhoA and PKA activity, respectively. Results from experiments with RhoA- and PKA-FRET sensors targeted to different subcellular compartments indicate that AMPH-elicited PKA activation occurs throughout the cell, whereas G13-mediated RhoA activation is concentrated near the endoplasmic reticulum. These observations define TAAR1 as an obligate intracellular target for amphetamines in dopamine neurons and support a model in which distinct pools of TAAR1 mediate the activation of signaling pathways in different compartments to regulate excitatory and dopaminergic neurotransmission.

Serotonin Transporter Ala276 Mouse: Novel Model to Assess the Neurochemical and Behavioral Impact of Thr276 Phosphorylation In Vivo.

The serotonin (5-HT) transporter (SERT) is a key regulator of 5-HT signaling and is a major target for antidepressants and psychostimulants. Human SERT coding variants have been identified in subjects with obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD) that impact transporter phosphorylation, cell surface trafficking and/or conformational dynamics. Prior to an initial description of a novel mouse line expressing the non-phosphorylatable SERT substitution Thr276Ala, we review efforts made to elucidate the structure and conformational dynamics of SERT with a focus on research implicating phosphorylation at Thr276 as a determinant of SERT conformational dynamics. Using the high-resolution structure of human SERT in inward- and outward-open conformations, we explore the conformation dependence of SERT Thr276 exposure, with results suggesting that phosphorylation is likely restricted to an inward-open conformation, consistent with prior biochemical studies. Assessment of genotypes from SERT/Ala276 heterozygous matings revealed a deviation from Mendelian expectations, with reduced numbers of Ala276 offspring, though no genotype differences were seen in growth or physical appearance. Similarly, no genotype differences were evident in midbrain or hippocampal 5-HT levels, midbrain and hippocampal SERT mRNA or midbrain protein levels, nor in midbrain synaptosomal 5-HT uptake kinetics. Behaviorally, SERT Ala276 homozygotes appeared normal in measures of anxiety and antidepressant-sensitive stress coping behavior. However, these mice displayed sex-dependent alterations in repetitive and social interactions, consistent with circuit-dependent requirements for Thr276 phosphorylation underlying these behaviors. Our findings indicate the utility of SERT Ala276 mice in evaluation of developmental, functional and behavioral consequences of regulatory SERT phosphorylation in vivo.

Variants of human CLDN9 cause mild to profound hearing loss.

Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis-interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi-allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

Structural models of the NaPi-II sodium-phosphate cotransporters.

Progress towards understanding the molecular mechanisms of phosphate homeostasis through sodium-dependent transmembrane uptake has long been stymied by the absence of structural information about the NaPi-II sodium-phosphate transporters. For many other coupled transporters, even those unrelated to NaPi-II, internal repeated elements have been revealed as a key feature that is inherent to their function. Here, we review recent structure prediction studies for NaPi-II transporters. Attempts to identify structural templates for NaPi-II transporters have leveraged the structural repeat perspective to uncover an otherwise obscured relationship with the dicarboxylate-sodium symporters (DASS). This revelation allowed the prediction of three-dimensional structural models of human NaPi-IIa and flounder NaPi-IIb, whose folds were evaluated by comparison with available biochemical data outlining the transmembrane topology and solvent accessibility of various regions of the protein. Using these structural models, binding sites for sodium and phosphate were proposed. The predicted sites were tested and refined based on detailed electrophysiological and biochemical studies and were validated by comparison with subsequently reported structures of transporters belonging to the AbgT family. Comparison with the DASS transporter VcINDY suggested a conformational mechanism involving a large, two-domain structural change, known as an elevator-like mechanism. These structural models provide a foundation for further studies into substrate binding, conformational change, kinetics, and energetics of sodium-phosphate transport. We discuss future opportunities, as well as the challenges that remain.

Cation Interactions and Membrane Potential Induce Conformational Changes in NaPi-IIb.

Voltage-dependence of Na(+)-coupled phosphate cotransporters of the SLC34 family arises from displacement of charges intrinsic to the protein and the binding/release of one Na(+) ion in response to changes in the transmembrane electric field. Candidate coordination residues for the cation at the Na1 site were previously predicted by structural modeling using the x-ray structure of dicarboxylate transporter VcINDY as template and confirmed by functional studies. Mutations at Na1 resulted in altered steady-state and presteady-state characteristics that should be mirrored in the conformational changes induced by membrane potential changes. To test this hypothesis by functional analysis, double mutants of the flounder SLC34A2 protein were constructed that contain one of the Na1-site perturbing mutations together with a substituted cysteine for fluorophore labeling, as expressed in Xenopus oocytes. The locations of the mutations were mapped onto a homology model of the flounder protein. The effects of the mutagenesis were characterized by steady-state, presteady-state, and fluorometric assays. Changes in fluorescence intensity (ΔF) in response to membrane potential steps were resolved at three previously identified positions. These fluorescence data corroborated the altered presteady-state kinetics upon perturbation of Na1, and furthermore indicated concomitant changes in the microenvironment of the respective fluorophores, as evidenced by changes in the voltage dependence and time course of ΔF. Moreover, iodide quenching experiments indicated that the aqueous nature of the fluorophore microenvironment depended on the membrane potential. These findings provide compelling evidence that membrane potential and cation interactions induce significant large-scale structural rearrangements of the protein.

Role of N-glycosylation in renal betaine transport.

The osmolyte and folding chaperone betaine is transported by the renal Na(+)-coupled GABA (γ-aminobutyric acid) symporter BGT-1 (betaine/GABA transporter 1), a member of the SLC6 (solute carrier 6) family. Under hypertonic conditions, the transcription, translation and plasma membrane (PM) insertion of BGT-1 in kidney cells are significantly increased, resulting in elevated betaine and GABA transport. Re-establishing isotonicity involves PM depletion of BGT-1. The molecular mechanism of the regulated PM insertion of BGT-1 during changes in osmotic stress is unknown. In the present study, we reveal a link between regulated PM insertion and N-glycosylation. Based on homology modelling, we identified two sites (Asn(171) and Asn(183)) in the extracellular loop 2 (EL2) of BGT-1, which were investigated with respect to trafficking, insertion and transport by immunogold-labelling, electron microscopy (EM), mutagenesis and two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radiolabelled substrate into MDCK (Madin-Darby canine kidney) and HEK293 (human embryonic kidney) cells. Trafficking and PM insertion of BGT-1 was clearly promoted by N-glycosylation in both oocytes and MDCK cells. Moreover, association with N-glycans at Asn(171) and Asn(183) contributed equally to protein activity and substrate affinity. Substitution of Asn(171) and Asn(183) by aspartate individually caused no loss of BGT-1 activity, whereas the double mutant was inactive, suggesting that N-glycosylation of at least one of the sites is required for function. Substitution by alanine or valine at either site caused a dramatic loss in transport activity. Furthermore, in MDCK cells PM insertion of N183D was no longer regulated by osmotic stress, highlighting the impact of N-glycosylation in regulation of this SLC6 transporter.

Identification of the first sodium binding site of the phosphate cotransporter NaPi-IIa (SLC34A1).

Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (Pi) in renal and intestinal epithelia. The transport cycle requires three Na(+) ions and one divalent Pi to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na(+) ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na(+) ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li(+) ions substitute for Na(+) at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and Pi based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and (32)P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li(+) ions to substitute for Na(+) ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.

Structural fold and binding sites of the human Na⁺-phosphate cotransporter NaPi-II.

Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in Pi homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na(+)-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na(+) and Pi coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.

Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter.

Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein-protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional (1)H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the "rocking bundle" hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.

Tentonin 3 is a pore-forming subunit of a slow inactivation mechanosensitive channel.

Mechanically activating (MA) channels transduce numerous physiological functions. Tentonin 3/TMEM150C (TTN3) confers MA currents with slow inactivation kinetics in somato- and barosensory neurons. However, questions were raised about its role as a Piezo1 regulator and its potential as a channel pore. Here, we demonstrate that purified TTN3 proteins incorporated into the lipid bilayer displayed spontaneous and pressure-sensitive channel currents. These MA currents were conserved across vertebrates and differ from Piezo1 in activation threshold and pharmacological response. Deep neural network structure prediction programs coupled with mutagenetic analysis predicted a rectangular-shaped, tetrameric structure with six transmembrane helices and a pore at the inter-subunit center. The putative pore aligned with two helices of each subunit and had constriction sites whose mutations changed the MA currents. These findings suggest that TTN3 is a pore-forming subunit of a distinct slow inactivation MA channel, potentially possessing a tetrameric structure.

Insights on the acetylated NF-κB transcription factor complex with DNA from molecular dynamics simulations.

The nuclear factor-κB (NF-κB) is a DNA sequence-specific regulator of many important biological processes, whose activity is modulated by enzymatic acetylation. In one of the best functionally characterized NF-κB complexes, the p50/p65 heterodimer, acetylation of K221 at p65 causes a decrease of DNA dissociation rate, whilst the acetylation of K122 and K123, also at p65, markedly decreases the binding affinity for DNA. By means of molecular dynamics simulations based on the X-ray structure of the p50/p65 complex with DNA, we provide insights on the structural determinants of the acetylated complexes in aqueous solution. Lysine acetylation involves the loss of favorable electrostatic interactions between DNA and NF-κB, which is partially compensated by the reduction of the desolvation free-energy of the two binding partners. Acetylation at both positions K122 and K123 is associated with a decrease of the electrostatic potential at the p65/DNA interface, which is only partially counterbalanced by an increase of the local Na(+) concentration. It induces the disruption of base-specific and nonspecific interactions between DNA and NF-κB and it is consistent with the observed decrease of binding affinity. In contrast, acetylation at position K221 results in the loss of nonspecific protein-DNA interactions, but the DNA recognition sites are not affected. In addition, the loss of protein-DNA interactions is likely to be counterbalanced by an increase of the configurational entropy of the complex, which provides, at a speculative level, a justification for the observed decrease of NF-κB/DNA dissociation rate.

Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.

Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia.

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

PLD2-PI(4,5)P2 interactions in fluid phase membranes: Structural modeling and molecular dynamics simulations.

Interaction of phospholipase D2 (PLD2) with phosphatidylinositol (4,5)-bisphosphate (PIP2) is regarded as the critical step of numerous physiological processes. Here we build a full-length model of human PLD2 (hPLD2) combining template-based and ab initio modeling techniques and use microsecond all-atom molecular dynamics (MD) simulations of the protein in contact with a complex membrane to determine hPLD2-PIP2 interactions. MD simulations reveal that the intermolecular interactions preferentially occur between specific PIP2 phosphate groups and hPLD2 residues; the most strongly interacting residues are arginine at the pbox consensus sequence (PX) and pleckstrin homology (PH) domain. Interaction networks indicate formation of clusters at the protein-membrane interface consisting of amino acids, PIP2, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA); the largest cluster was in the PH domain.

Mouse Models of Human Pathogenic Variants of TBC1D24 Associated with Non-Syndromic Deafness DFNB86 and DFNA65 and Syndromes Involving Deafness.

Human pathogenic variants of TBC1D24 are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human TBC1D24 associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of TBC1D24 c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea, Tbc1d24 expression was detected only in spiral ganglion neurons. We engineered mouse models of DFNB86 p.(Asp70Tyr) and DFNA65 p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human TBC1D24. Unexpectedly, no auditory dysfunction was detected in Tbc1d24 mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse Tbc1d24 and human TBC1D24.