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Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.
Foegeding NJ, Raghunathan K, Campbell AM, Kim SW, Lau KS, Kenworthy AK, Cover TL, Ohi MD
(2019) Infect Immun 87:
MeSH Terms: Autophagy, Bacterial Proteins, Cell Line, Cell Survival, Epithelial Cells, Gastric Mucosa, Helicobacter Infections, Helicobacter pylori, Humans, Hydrogen-Ion Concentration, Muramidase, Protein Stability, Protein Transport, Proteolysis
Show Abstract · Added February 7, 2019
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.
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14 MeSH Terms
Dodecyl-β-melibioside Detergent Micelles as a Medium for Membrane Proteins.
Hutchison JM, Lu Z, Li GC, Travis B, Mittal R, Deatherage CL, Sanders CR
(2017) Biochemistry 56: 5481-5484
MeSH Terms: Amyloid beta-Protein Precursor, Detergents, Diacylglycerol Kinase, Disaccharides, Dynamic Light Scattering, Enzyme Stability, Escherichia coli Proteins, Glucosides, Glycolipids, Hot Temperature, Humans, Micelles, Myelin Proteins, Nuclear Magnetic Resonance, Biomolecular, Particle Size, Peptide Fragments, Protein Interaction Domains and Motifs, Protein Stability, Receptor, Notch1
Show Abstract · Added November 21, 2018
There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the β-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into β-DDMB yielded activities that were 40% higher than those of DAGK purified into β-DDM. β-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. β-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.
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MeSH Terms
Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.
Gaskill CF, Carrier EJ, Kropski JA, Bloodworth NC, Menon S, Foronjy RF, Taketo MM, Hong CC, Austin ED, West JD, Means AL, Loyd JE, Merryman WD, Hemnes AR, De Langhe S, Blackwell TS, Klemm DJ, Majka SM
(2017) J Clin Invest 127: 2262-2276
MeSH Terms: ATP Binding Cassette Transporter, Subfamily G, Member 2, Animals, Bone Morphogenetic Protein Receptors, Type II, Cell Differentiation, Cell Lineage, Cells, Cultured, Humans, Lung, Mesenchymal Stem Cells, Mice, Transgenic, Microvessels, Neovascularization, Pathologic, Pericytes, Protein Stability, Pulmonary Fibrosis, Vasoconstriction, Wnt Signaling Pathway
Show Abstract · Added June 6, 2017
Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.
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17 MeSH Terms
Structure-Based Design of a Soluble Prefusion-Closed HIV-1 Env Trimer with Reduced CD4 Affinity and Improved Immunogenicity.
Chuang GY, Geng H, Pancera M, Xu K, Cheng C, Acharya P, Chambers M, Druz A, Tsybovsky Y, Wanninger TG, Yang Y, Doria-Rose NA, Georgiev IS, Gorman J, Joyce MG, O'Dell S, Zhou T, McDermott AB, Mascola JR, Kwong PD
(2017) J Virol 91:
MeSH Terms: AIDS Vaccines, Animals, Antibodies, Neutralizing, CD4 Antigens, Guinea Pigs, HIV Antibodies, HIV Antigens, HIV-1, Humans, Protein Multimerization, Protein Stability, env Gene Products, Human Immunodeficiency Virus
Show Abstract · Added May 3, 2017
The HIV-1 envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and coreceptor-bound conformations by transitioning into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1 Env trimer to its prefusion-closed state as this state is recognized by most broadly neutralizing, but not nonneutralizing, antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env trimer mimic developed by Sanders, Moore, and colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-Å resolution and of an additional DS-SOSIP.6mut variant at 4.3-Å resolution, and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier 2 autologous titers versus tier 1 V3-sensitive titers than BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggest utility as an immunogen or a serologic probe; moreover, the specific four alterations identified here, M154, M300, M302, and L320 (4mut), can also be transferred to other HIV-1 Env trimers of interest to improve their properties. One approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the structurally flexible HIV-1 envelope (Env) trimer in a conformation that displays predominantly broadly neutralizing epitopes and few to no nonneutralizing epitopes. The prefusion-closed conformation of HIV-1 Env has been identified as one such preferred conformation, and a current leading vaccine candidate is the BG505 DS-SOSIP variant, comprising two disulfides and an Ile-to-Pro mutation of Env from strain BG505. Here, we introduced additional mutations to further stabilize BG505 DS-SOSIP in the vaccine-preferred prefusion-closed conformation. In guinea pigs, our best mutant, DS-SOSIP.4mut, elicited a significantly higher ratio of autologous versus V3-directed neutralizing antibody responses than the SOSIP-stabilized form. We also observed an improvement in thermostability and a reduction in CD4 affinity. With improved antigenicity, stability, and immunogenicity, DS-SOSIP.4mut-stabilized trimers may have utility as HIV-1 immunogens or in other antigen-specific contexts, such as with B-cell probes.
Copyright © 2017 American Society for Microbiology.
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12 MeSH Terms
SKP2 loss destabilizes EZH2 by promoting TRAF6-mediated ubiquitination to suppress prostate cancer.
Lu W, Liu S, Li B, Xie Y, Izban MG, Ballard BR, Sathyanarayana SA, Adunyah SE, Matusik RJ, Chen Z
(2017) Oncogene 36: 1364-1373
MeSH Terms: Animals, Cell Line, Tumor, Disease Models, Animal, Enhancer of Zeste Homolog 2 Protein, Gene Knockout Techniques, Histones, Humans, Lysine, Male, Mice, PTEN Phosphohydrolase, Prostate, Prostatic Neoplasms, Protein Stability, S-Phase Kinase-Associated Proteins, TNF Receptor-Associated Factor 6, Tumor Suppressor Protein p53, Ubiquitination
Show Abstract · Added April 18, 2017
EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.
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18 MeSH Terms
Documentation of an Imperative To Improve Methods for Predicting Membrane Protein Stability.
Kroncke BM, Duran AM, Mendenhall JL, Meiler J, Blume JD, Sanders CR
(2016) Biochemistry 55: 5002-9
MeSH Terms: Membrane Proteins, Point Mutation, Protein Stability, Thermodynamics
Show Abstract · Added April 7, 2017
There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (ΔΔG < -0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins.
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4 MeSH Terms
Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV.
Joyce MG, Zhang B, Ou L, Chen M, Chuang GY, Druz A, Kong WP, Lai YT, Rundlet EJ, Tsybovsky Y, Yang Y, Georgiev IS, Guttman M, Lees CR, Pancera M, Sastry M, Soto C, Stewart-Jones GBE, Thomas PV, Van Galen JG, Baxa U, Lee KK, Mascola JR, Graham BS, Kwong PD
(2016) Nat Struct Mol Biol 23: 811-820
MeSH Terms: Animals, Crystallography, X-Ray, Female, Glycoproteins, HEK293 Cells, Humans, Male, Mice, Inbred C57BL, Models, Molecular, Protein Engineering, Protein Stability, Respiratory Syncytial Virus Infections, Respiratory Syncytial Viruses, Vaccination, Viral Fusion Proteins, Viral Vaccines
Show Abstract · Added May 3, 2017
Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.
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16 MeSH Terms
Identification of a Paralog-Specific Notch1 Intracellular Domain Degron.
Broadus MR, Chen TW, Neitzel LR, Ng VH, Jodoin JN, Lee LA, Salic A, Robbins DJ, Capobianco AJ, Patton JG, Huppert SS, Lee E
(2016) Cell Rep 15: 1920-9
MeSH Terms: Amino Acid Sequence, Animals, Cell Extracts, Embryo, Nonmammalian, F-Box Proteins, HEK293 Cells, Humans, Muscle Proteins, Mutation, Protein Binding, Protein Domains, Protein Stability, Proteolysis, Receptor, Notch1, Regulatory Sequences, Nucleic Acid, Sequence Homology, Amino Acid, Transcription, Genetic, Ubiquitin-Protein Ligases, Xenopus, Zebrafish
Show Abstract · Added February 13, 2017
Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism by which degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.
Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
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20 MeSH Terms
Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase.
Wang Z, Tacchelly-Benites O, Yang E, Thorne CA, Nojima H, Lee E, Ahmed Y
(2016) Genetics 203: 269-81
MeSH Terms: Adenomatous Polyposis Coli Protein, Animals, Axin Protein, Drosophila, Genotype, Mitosis, Protein Interaction Domains and Motifs, Protein Stability, Tankyrases, Wnt Proteins, Wnt Signaling Pathway, Xenopus
Show Abstract · Added February 13, 2017
Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein "destruction complex," assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three- to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation.
Copyright © 2016 by the Genetics Society of America.
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12 MeSH Terms
LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding.
Layer JH, Alford CE, McDonald WH, Davé UP
(2016) Mol Cell Biol 36: 488-506
MeSH Terms: Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Amino Acid Substitution, Cell Line, DNA-Binding Proteins, Humans, Jurkat Cells, LIM Domain Proteins, Leukemia, T-Cell, Molecular Sequence Data, Mutation, Protein Interaction Domains and Motifs, Protein Interaction Maps, Protein Stability, Proto-Oncogene Proteins, Transcription Factors, Transcriptional Activation
Show Abstract · Added January 26, 2016
LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R(320)LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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17 MeSH Terms