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- Structural basis for neutralization of cytotoxic abrin by monoclonal antibody D6F10. [Journal Article]
- FJFEBS J 2018 Dec 06
- Abrin, an extremely cytotoxic Type-II ribosome inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28...
Abrin, an extremely cytotoxic Type-II ribosome inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28S rRNA thereby arresting protein synthesis and leading to cell death. Monoclonal antibody (mAb) D6F10 is the only known antibody that neutralizes ABA's activity in cell-free systems as well as abrin's toxicity in vitro and in vivo. However, how binding of mAb D6F10 to abrin interferes with abrin's catalytic activity at ribosome is still poorly understood. To provide structural basis for mAb D6F10-mediated rescue of ribosome inactivation by abrin, we determined crystal structures of ABA with and without substrate analogs. The structures of ABA-substrate analogs and ribosome were used in an experiment-guided computational protocol, to construct the ABA-Ribosome complex. A homology model of the variable region (Fv ) of mAb D6F10 was generated and docked with the apo-ABA structure to construct the ABA-D6F10 Fv complex. Structural superposition of ABA common to ABA-D6F10 Fv and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. In contrast to ABA alone, ABA bound to mAb D6F10 is unable to access the SRL on the ribosome owing to steric clashes of mAb D6F10 with the ribosome. Crystal structures of ABA also reveal a catalytic water molecule implicated in hydrolyzing N-glycosidic bond of the susceptible adenosine by RIPs. Furthermore, our strategy can be used to provide structural details of steric hindrance important for neutralization of ricin, another RIP, by mAb 6C2 and hence is of wide applicability. This article is protected by copyright. All rights reserved.
- Interplay of Cysteine Exposure and Global Protein Dynamics in Small-molecule Recognition by a Regulator of G-protein Signaling Protein. [Journal Article]
- PProteins 2018 Dec 06
- Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein-coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic targe...
Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein-coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic target. Recently discovered thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for the RGS4 protein, thereby suggesting intrinsic differences in dynamics of this protein upon binding of these compounds. In this work, we investigated using atomistic molecular dynamics (MD) simulations the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, we studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. We found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. We further measured inhibitory effects of TDZD compounds using a protein-protein interaction assay on a single-cysteine RGS4 protein showing trends in potencies of compounds consistent with our simulation studies. Thermodynamic analyses of RGS4 conformations in the apo-state and on binding to TDZD compounds revealed links between both conformational models of RGS4. The exposure of cysteine side-chains appears to facilitate initial binding of TDZD compounds followed by migration of the compound into a bundle of four helices, thereby causing allosteric perturbations of the RGS/Gα protein-protein interface. This article is protected by copyright. All rights reserved.
- Hematopoietic Progenitor Kinase-1 Structure in a Domain-Swapped Dimer. [Journal Article]
- SStructure 2018 Nov 13
- Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator ...
Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.
- Lipoprotein-X fifty years after its original discovery. [Review]
- NMNutr Metab Cardiovasc Dis 2018 Sep 26
- CONCLUSIONS: Lp-X might be considered a defense mechanism against the toxic effect of free cholesterol in cholestasis. The frequency of cardiovascular events in patients affected by primary biliary cholangitis, in whom the Lp-X is present in high concentration, are not increased. Further studies could now clarify the remaining open questions on the role of Lp-X in the dyslipidemia of cholestasis.
- Crystal structures of arginine sensor CASTOR1 in arginine-bound and ligand free states. [Journal Article]
- BBBiochem Biophys Res Commun 2018 Nov 28
- The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a master regulator of metabolism and cell growth. Among the numerous extracellular and intracellular signals, certain amino acids acti...
The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a master regulator of metabolism and cell growth. Among the numerous extracellular and intracellular signals, certain amino acids activate mTORC1 in a Rag-dependent manner. Arginine can stimulate mTORC1 activity by releasing the inhibitor CASTOR1 (Cellular Arginine Sensor of mTORC1) from GATOR2, a positive regulator of mTORC1 which interacts with GATOR1, the GAP for RagA/B. Three groups have resolved the structures of arginine-CASTOR1 complex, shedding a new light on molecular basis of the regulation of mTORC1 activity by arginine. However, lacking the apo structure of CASTOR1 prelimited the molecular understanding of mechanism underlying mTORC1 regulation. Here, we report crystal structures of arginine sensor CASTOR1 in arginine-bound and ligand free states at 2.05 Å and 2.8 Å, respectively. Structural comparison of CASTOR1 between two states reveals near identical conformations, except in two loop regions. It indicates CASTOR1 does not undergo large conformational change during arginine binding. Therefore, we conclude a detailed structural interpretation of arginine sensing by CASTOR1 in mTORC1 pathway.
- Improving the Accuracy of Protein-Ligand Binding Mode Prediction Using a Molecular Dynamics-Based Pocket Generation Approach. [Journal Article]
- JCJ Comput Chem 2018 Dec 15; 39(32):2679-2689
- Protein-drug binding mode prediction from the apo-protein structure is challenging because drug binding often induces significant protein conformational changes. Here, the authors report a computatio...
Protein-drug binding mode prediction from the apo-protein structure is challenging because drug binding often induces significant protein conformational changes. Here, the authors report a computational workflow that incorporates a novel pocket generation method. First, the closed protein pocket is expanded by repeatedly filling virtual atoms during molecular dynamics (MD) simulations. Second, after ligand docking toward the prepared pocket structures, binding mode candidates are ranked by MD/Molecular Mechanics Poisson-Boltzmann Surface Area. The authors validated our workflow using CDK2 kinase, which has an especially-closed ATP-binding pocket in the apo-form, and several inhibitors. The crystallographic pose coincided with the top-ranked docking pose for 59% (34/58) of the compounds and was within the top five-ranked ones for 88% (51/58), while those estimated by a conventional prediction protocol were 9% (5/58) and 50% (29/58), respectively. Our study demonstrates that the prediction accuracy is significantly improved by preceding pocket expansion, leading to generation of conformationally-diverse binding mode candidates. © 2018 Wiley Periodicals, Inc.
- Substrate polyspecificity and conformational relevance in ABC transporters: new insights from structural studies. [Review]
- BSBiochem Soc Trans 2018 Dec 04
- Transport of molecules and ions across biological membranes is an essential process in all organisms. It is carried out by a range of evolutionarily conserved primary and secondary transporters. A si...
Transport of molecules and ions across biological membranes is an essential process in all organisms. It is carried out by a range of evolutionarily conserved primary and secondary transporters. A significant portion of the primary transporters belong to the ATP-binding cassette (ABC) superfamily, which utilise the free-energy from ATP hydrolysis to shuttle many different substrates across various biological membranes, and consequently, are involved in both normal and abnormal physiology. In humans, ABC transporter-associated pathologies are perhaps best exemplified by multidrug-resistance transporters that efflux many xenobiotic compounds due to their remarkable substrate polyspecificity. Accordingly, understanding the transport mechanism(s) is of great significance, and indeed, much progress has been made in recent years, particularly from structural studies on ABC exporters. Consequently, the general mechanism of 'alternate access' has been modified to describe individual transporter nuances, though some aspects of the transport process remain unclear. Moreover, as new information has emerged, the physiological relevance of the 'open-apo' conformation of MsbA (a bacterial exporter) has been questioned and, by extension, its contribution to mechanistic models. We present here a comprehensive overview of the most recently solved structures of ABC exporters, focusing on new insights regarding the nature of substrate polyspecificity and the physiological relevance of the 'open-apo' conformation. This review evaluates the claim that the latter may be an artefact of detergent solubilisation, and we hypothesise that the biophysical properties of the membrane play a key role in the function of ABC exporters allowing them to behave like a 'spring-hinge' during their transport cycle.
- Prognostic Significance of Pretreatment Apolipoprotein A-I as a Noninvasive Biomarker in Cancer Survivors: A Meta-Analysis. [Review]
- DMDis Markers 2018; 2018:1034037
- Numerous studies have reported the prognostic significance of serum apolipoprotein A-I (ApoA-I) in various cancers, but the results have been inconsistent. The current meta-analysis was performed to ...
Numerous studies have reported the prognostic significance of serum apolipoprotein A-I (ApoA-I) in various cancers, but the results have been inconsistent. The current meta-analysis was performed to investigate the association between ApoA-I level and prognosis in human malignancies.
- Establishment, validation and application of a New World Primate model of ETEC disease for vaccine development. [Journal Article]
- IIInfect Immun 2018 Dec 03
- The establishment of an animal model that closely approximates ETEC disease in humans is critical for the development and evaluation of vaccines against this enteropathogen. Here, we evaluated the su...
The establishment of an animal model that closely approximates ETEC disease in humans is critical for the development and evaluation of vaccines against this enteropathogen. Here, we evaluated the susceptibility of Aotus nancymaae, a New World monkey species, to ETEC infection. Animals were challenged orogastrically with 109-1011 colony forming units (CFU) of the human pathogenic CFA/I+ ETEC strain H10407 and examined for evidence of diarrhea and fecal shedding of bacteria. A clear dose range effect was obtained with diarrheal attack rates of 40%- 80%, validated in a follow on study demonstrating an attack rate of 80% with 1011 CFU of H10407 ETEC. To determine whether this model is an effective approach for assessing ETEC vaccine candidates, we used it to evaluate the ability of the donor strand-complemented CFA/I adhesin CfaE (dscCfaE) to protect against H10407 challenge. In a series of experiments, animals were vaccinated with dscCfaE alone, dscCfaE with either CTB or LTB, or PBS alone and then challenged with 1011 CFU of H10407. Control animals vaccinated with PBS had attack rates of 70-90% on challenge. Vaccination with dscCfaE, or dscCfaE admixed with CTB or LTB resulted in a reduction of attack rates, with vaccine efficacies of 66.7% (P=0.02), 77.7% (P=0.006) and 42.9% (P=0.370) to 83.3% (P=0.041), respectively. In conclusion, we have shown the H10407 ETEC challenge of A. nancymaae to be an effective, reproducible model of ETEC disease and importantly, have demonstrated that in this model vaccination with the prototype vaccine candidate dscCfaE is protective against CF-homologous disease.
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- Preoperative apolipoprotein B/A1 ratio is an independent prognostic factor in metastatic renal cell carcinoma. [Journal Article]
- UOUrol Oncol 2018 Nov 30
- CONCLUSIONS: Preoperative Apo B/A1 ratio is an independent prognostic factor for PFS and OS in patients with mRCC. Preoperative Apo B/A1 ratio can be useful in improving current prognostic evaluation and treatment decision for patients with mRCC.