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colonizes the gastric mucosa and secretes a pore-forming toxin (VacA). Two main types of VacA, m1 and m2, can be distinguished by phylogenetic analysis. Type m1 forms of VacA have been extensively studied, but there has been relatively little study of m2 forms. In this study, we generated strains producing chimeric proteins in which VacA m1 segments of a parental strain were replaced by corresponding m2 sequences. In comparison to the parental m1 VacA protein, a chimeric protein (designated m2/m1) containing m2 sequences in the N-terminal portion of the m region was less potent in causing vacuolation of HeLa cells, AGS gastric cells, and AZ-521 duodenal cells and had reduced capacity to cause membrane depolarization or death of AZ-521 cells. Consistent with the observed differences in activity, the chimeric m2/m1 VacA protein bound to cells at reduced levels compared to the binding levels of the parental m1 protein. The presence of two strain-specific insertions or deletions within or adjacent to the m region did not influence toxin activity. Experiments with human gastric organoids grown as monolayers indicated that m1 and m2/m1 forms of VacA had similar cell-vacuolating activities. Interestingly, both forms of VacA bound preferentially to the basolateral surface of organoid monolayers and caused increased cell vacuolation when interacting with the basolateral surface compared to the apical surface. These data provide insights into functional correlates of sequence variation in the VacA midregion (m region).
Copyright © 2020 American Society for Microbiology.
Metazoan retromer (VPS26/VPS35/VPS29) associates with sorting nexins on endosomal tubules to sort proteins to the trans-Golgi network or plasma membrane. Mechanisms of metazoan retromer assembly remain undefined. We combine single-particle cryoelectron microscopy with biophysical methods to uncover multiple oligomer structures. 2D class averages reveal mammalian heterotrimers; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical solution studies. We provide reconstructions of all species, including key sub-structures (∼5 Å resolution). Local resolution variation suggests that heterotrimers and dimers adopt multiple conformations. Our structures identify a flexible, highly conserved electrostatic dimeric interface formed by VPS35 subunits. We generate structure-based mutants to disrupt this interface in vitro. Equivalent mutations in yeast demonstrate a mild cargo-sorting defect. Our data suggest the metazoan retromer is an adaptable and plastic scaffold that accommodates interactions with different sorting nexins to sort multiple cargoes from endosomes their final destinations.
Copyright © 2020 Elsevier Ltd. All rights reserved.
Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.
In the brain, AMPA-type glutamate receptors (AMPARs) form complexes with their auxiliary subunits and mediate the majority of fast excitatory neurotransmission. Signals transduced by these complexes are critical for synaptic plasticity, learning, and memory. The two major categories of AMPAR auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs) and cornichon homologs (CNIHs); these subunits share little homology and play distinct roles in controlling ion channel gating and trafficking of AMPAR. Here, I report high-resolution cryo-electron microscopy structures of AMPAR in complex with CNIH3. Contrary to its predicted membrane topology, CNIH3 lacks an extracellular domain and instead contains four membrane-spanning helices. The protein-protein interaction interface that dictates channel modulation and the lipids surrounding the complex are revealed. These structures provide insights into the molecular mechanism for ion channel modulation and assembly of AMPAR/CNIH3 complexes.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
BACKGROUND - Mutations in SLC6A1 have been associated mainly with myoclonic atonic epilepsy (MAE) and intellectual disability. We identified a novel missense mutation in a patient with Lennox-Gastaut syndrome (LGS) characterized by severe seizures and developmental delay.
METHODS - Exome Sequencing was performed in an epilepsy patient cohort. The impact of the mutation was evaluated by H γ-aminobutyric acid (GABA) uptake, structural modeling, live cell microscopy, cell surface biotinylation and a high-throughput assay flow cytometry in both neurons and non neuronal cells.
RESULTS - We discovered a heterozygous missense mutation (c700G to A [pG234S) in the SLC6A1 encoding GABA transporter 1 (GAT-1). Structural modeling suggests the mutation destabilizes the global protein conformation. With transient expression of enhanced yellow fluorescence protein (YFP) tagged rat GAT-1 cDNAs, we demonstrated that the mutant GAT-1(G234S) transporter had reduced total protein expression in both rat cortical neurons and HEK 293 T cells. With a high-throughput flow cytometry assay and live cell surface biotinylation, we demonstrated that the mutant GAT-1(G234S) had reduced cell surface expression. H radioactive labeling GABA uptake assay in HeLa cells indicated a reduced function of the mutant GAT-1(G234S).
CONCLUSIONS - This mutation caused instability of the mutant transporter protein, which resulted in reduced cell surface and total protein levels. The mutation also caused reduced GABA uptake in addition to reduced protein expression, leading to reduced GABA clearance, and altered GABAergic signaling in the brain. The impaired trafficking and reduced GABA uptake function may explain the epilepsy phenotype in the patient.
Copyright © 2019. Published by Elsevier Inc.
Allosteric regulation of methylmalonyl-CoA mutase (MCM) by the G-protein chaperone CblA is transduced via three "switch" elements that gate the movement of the B cofactor to and from MCM. Mutations in CblA and MCM cause hereditary methylmalonic aciduria. Unlike the bacterial orthologs used previously to model disease-causing mutations, human MCM and CblA exhibit a complex pattern of regulation that involves interconverting oligomers, which are differentially sensitive to the presence of GTP versus GDP. Patient mutations in the switch III region of CblA perturb the nucleotide-sensitive distribution of the oligomeric complexes with MCM, leading to loss of regulated movement of B to and/or from MCM and explain the molecular mechanism of the resulting disease.
Copyright © 2019 Elsevier Ltd. All rights reserved.
The classic mode of G protein-coupled receptor (GPCR)-mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)-mediated cleavage of plasma membrane-anchored EGFR ligands. Herein, we show that the Gαs-activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E (PGE ) transactivate EGFR through increased cell-surface delivery of the EGFR ligand transforming growth factor-α (TGFα) in polarizing madin-darby canine kidney (MDCK) and Caco-2 cells. This is achieved by PKA-mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα-containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser-223, a process that is facilitated by the molecular scaffold A-kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell-surface delivery of TGFα and increased EGFR activation. Thus, GPCR-triggered, PKA/AKAP12/NKD2-regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.
© 2019 The Authors. Traffic published by John Wiley & Sons Ltd.
Na-K-2Cl cotransporter-1 (NKCC1) mediates the electroneutral transport of Na, K, and Cl and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.
VacA is a secreted pore-forming toxin that induces cell vacuolation and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We observed that purified VacA has relatively little effect on the viability of AGS gastric epithelial cells, but the presence of exogenous weak bases such as ammonium chloride (NHCl) enhances the susceptibility of these cells to VacA-induced vacuolation and cell death. Therefore, we tested the hypothesis that NHCl augments VacA toxicity by altering the intracellular trafficking of VacA or inhibiting intracellular VacA degradation. We observed VacA colocalization with LAMP1- and LC3-positive vesicles in both the presence and absence of NHCl, indicating that NHCl does not alter VacA trafficking to lysosomes or autophagosomes. Conversely, we found that supplemental NHCl significantly increases the intracellular stability of VacA. By conducting experiments using chemical inhibitors, stable ATG5 knockdown cell lines, and ATG16L1 knockout cells (generated using CRISPR/Cas9), we show that VacA degradation is independent of autophagy and proteasome activity but dependent on lysosomal acidification. We conclude that weak bases like ammonia, potentially generated during infection by urease and other enzymes, enhance VacA toxicity by inhibiting toxin degradation.
Copyright © 2019 American Society for Microbiology.
Annexin proteins function as Ca-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.
© 2019 McCulloch et al.