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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.
is a Gram-negative opportunistic pathogen that causes diverse infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and acquired antimicrobial-resistance mechanisms, isolates are commonly multidrug resistant, and infections are notoriously difficult to treat. The World Health Organization recently highlighted carbapenem-resistant as a "critical priority" for the development of new antimicrobials because of the risk to human health posed by this organism. Therefore, it is important to discover the mechanisms used by to survive stresses encountered during infection in order to identify new drug targets. In this study, by use of imaging, we identified hydrogen peroxide (HO) as a stressor produced in the lung during infection and defined OxyR as a transcriptional regulator of the HO stress response. Upon exposure to HO, differentially transcribes several hundred genes. However, the transcriptional upregulation of genes predicted to detoxify hydrogen peroxide is abolished in an strain in which the transcriptional regulator is genetically inactivated. Moreover, inactivation of in both antimicrobial-susceptible and multidrug-resistant strains impairs growth in the presence of HO OxyR is a direct regulator of and , which encode the major HO-degrading enzymes in , as confirmed through measurement of promoter binding by recombinant OxyR in electromobility shift assays. Finally, an mutant is less fit than wild-type during infection of the murine lung. This work reveals a mechanism used by this important human pathogen to survive HO stress encountered during infection.
Copyright © 2018 American Society for Microbiology.
Esophageal adenocarcinoma (EAC) is a highly aggressive malignancy that is characterized by resistance to chemotherapy and a poor clinical outcome. The overexpression of the receptor tyrosine kinase AXL is frequently associated with unfavorable prognosis in EAC. Although it is well documented that AXL mediates cancer cell invasion as a downstream effector of epithelial-to-mesenchymal transition, the precise molecular mechanism underlying this process is not completely understood. Herein, we demonstrate for the first time that AXL mediates cell invasion through the regulation of lysosomes peripheral distribution and cathepsin B secretion in EAC cell lines. Furthermore, we show that AXL-dependent peripheral distribution of lysosomes and cell invasion are mediated by extracellular acidification, which is potentiated by AXL-induced secretion of lactate through AKT-NF-κB-dependent MCT-1 regulation. Our novel mechanistic findings support future clinical studies to evaluate the therapeutic potential of the AXL inhibitor R428 (BGB324) in highly invasive EAC.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
BACKGROUND & AIMS - Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters.
METHODS - We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCre;Myo5b mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry.
RESULTS - Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCre;Myo5b mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCre;Myo5b mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCre;Myo5b mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity.
CONCLUSIONS - Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.
Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.
Hyperkalemia in association with metabolic acidosis that are out of proportion to changes in glomerular filtration rate defines type 4 renal tubular acidosis (RTA), the most common RTA observed, but the molecular mechanisms underlying the associated metabolic acidosis are incompletely understood. We sought to determine whether hyperkalemia directly causes metabolic acidosis and, if so, the mechanisms through which this occurs. We studied a genetic model of hyperkalemia that results from early distal convoluted tubule (DCT)-specific overexpression of constitutively active Ste20/SPS1-related proline-alanine-rich kinase (DCT-CA-SPAK). DCT-CA-SPAK mice developed hyperkalemia in association with metabolic acidosis and suppressed ammonia excretion; however, titratable acid excretion and urine pH were unchanged compared with those in wild-type mice. Abnormal ammonia excretion in DCT-CA-SPAK mice associated with decreased proximal tubule expression of the ammonia-generating enzymes phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and overexpression of the ammonia-recycling enzyme glutamine synthetase. These mice also had decreased expression of the ammonia transporter family member Rhcg and decreased apical polarization of H-ATPase in the inner stripe of the outer medullary collecting duct. Correcting the hyperkalemia by treatment with hydrochlorothiazide corrected the metabolic acidosis, increased ammonia excretion, and normalized ammoniagenic enzyme and Rhcg expression in DCT-CA-SPAK mice. In wild-type mice, induction of hyperkalemia by administration of the epithelial sodium channel blocker benzamil caused hyperkalemia and suppressed ammonia excretion. Hyperkalemia decreases proximal tubule ammonia generation and collecting duct ammonia transport, leading to impaired ammonia excretion that causes metabolic acidosis.
Copyright © 2018 by the American Society of Nephrology.
Integrins are transmembrane cell-extracellular matrix adhesion receptors that impact many cellular functions. A subgroup of integrins contain an inserted (I) domain within the α-subunits (αI) that mediate ligand recognition where function is contingent on binding a divalent cation at the metal ion dependent adhesion site (MIDAS). Ca is reported to promote α1I but inhibit α2I ligand binding. We co-crystallized individual I-domains with MIDAS-bound Ca and report structures at 1.4 and 2.15 Å resolution, respectively. Both structures are in the "closed" ligand binding conformation where Ca induces minimal global structural changes. Comparisons with Mg-bound structures reveal Mg and Ca bind α1I in a manner sufficient to promote ligand binding. In contrast, Ca is displaced in the α2I domain MIDAS by 1.4 Å relative to Mg and unable to directly coordinate all MIDAS residues. We identified an E152-R192 salt bridge hypothesized to limit the flexibility of the α2I MIDAS, thus, reducing Ca binding. A α2I E152A construct resulted in a 10,000-fold increase in Mg and Ca binding affinity while increasing binding to collagen ligands 20%. These data indicate the E152-R192 salt bridge is a key distinction in the molecular mechanism of differential ion binding of these two I domains.
Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2 mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.
Copyright © 2018 Elsevier Inc. All rights reserved.
We have previously reported that the dispersion of spin-lattice relaxation rates in the rotating frame (R ) of tissue water protons at high field can be dominated by chemical exchange contributions. Ischemia in brain causes changes in tissue pH, which in turn may affect proton exchange rates. Amide proton transfer (APT, a form of chemical exchange saturation transfer) has been shown to be sensitive to chemical exchange rates and able to detect pH changes non-invasively following ischemic stroke. However, the specificity of APT to pH changes is decreased because of the influence of several other factors that affect magnetization transfer. R is less influenced by such confounding factors and thus may be more specific for detecting variations in pH. Here, we applied a spin-locking sequence to detect ischemic stroke in animal models. Although R images acquired with a single spin-locking amplitude (ω ) have previously been used to assess stroke, here we use ΔR , which is the difference in R values acquired with two different locking fields to emphasize selectively the contribution of chemical exchange effects. Numerical simulations with different exchange rates and measurements of tissue homogenates with different pH were performed to evaluate the specificity of ΔR to detect tissue acidosis. Spin-lock and APT data were acquired on five rat brains after ischemic strokes induced via middle cerebral artery occlusions. Correlations between these data were analyzed at different time points after the onset of stroke. The results show that ΔR (but not R acquired with a single ω ) was significantly correlated with APT metrics consistent with ΔR varying with pH.
Copyright © 2018 John Wiley & Sons, Ltd.
The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. (2017) 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.