The Experts below are selected from a list of 189585 Experts worldwide ranked by ideXlab platform

Howard Robinson - One of the best experts on this subject based on the ideXlab platform.

  • reversible s nitrosylation in an engineered azurin
    Nature Chemistry, 2016
    Co-Authors: Shiliang Tian, Jing Liu, Ryan E Cowley, Parisa Hosseinzadeh, Nicholas M Marshall, Howard Robinson, Mark J Nilges, Ninian J Blackburn, Edward I Solomon
    Abstract:

    S-Nitrosylation has emerged as an important pathway for dynamic Post-Translational Regulation of many classes of proteins. Now, the reversible insertion of NO into a copper–thiolate bond has been observed under physiologically relevant conditions using an engineered azurin. DFT calculation indicates that the reaction proceeds via a radical combination mechanism.

  • dimeric c di gmp is required for post translational Regulation of alginate production in pseudomonas aeruginosa
    Journal of Biological Chemistry, 2015
    Co-Authors: John C Whitney, Howard Robinson, Gregory B Whitfield, Lindsey S Marmont, Mirela A Neculai, Y D Lobsanov, Dennis E Ohman, Lynne P Howell
    Abstract:

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity Post-Translationally regulates alginate secretion. In this study, we report the 1.8 A crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor producedmore » by P. aeruginosa.« less

Steven C Huber - One of the best experts on this subject based on the ideXlab platform.

  • post translational Regulation of nitrate reductase mechanism physiological relevance and environmental triggers
    Journal of Experimental Botany, 2001
    Co-Authors: Werner M Kaiser, Steven C Huber
    Abstract:

    Assimilatory nitrate reductase (NR) of higher plants is a most interesting enzyme, both from its central function in plant primary metabolism and from the complex Regulation of its expression and control of catalytic activity and degradation. Here, present knowledge about the mechanism of Post-Translational Regulation of NR is summarized and the properties of the regulatory enzymes involved (protein kinases, protein phosphatases and 14-3-3-binding proteins) are described. It is shown that light and oxygen availability are the major external triggers for the rapid and reversible modulation of NR activity, and that sugars and or sugar phosphates are the internal signals which regulate the protein kinase(s) and phosphatase. It is also demonstrated that stress factors like nitrate deficiency and salinity have remarkably little direct influence on the NR activation state. Further, changes in NR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that NR can be under strong substrate limitation. The degradation and half-life of the NR protein also appear to be affected by NR phosphorylation and 14-3-3 binding, as NR activation always correlates positively with its stability. However, it is not known whether the molecular form of NR in vivo affects its susceptibility to proteolytic degradation, or whether factors that affect the NR activation state also independently affect the activity or induction of the NR protease(s). A second and potentially important function of NR, the production of nitric oxide (NO) from nitrite is briefly described, but it remains to be determined whether NR produces NO for pathogen stress signalling in vivo.

  • identification in vitro of a post translational regulatory site in the hinge 1 region of arabidopsis nitrate reductase
    The Plant Cell, 1996
    Co-Authors: Steven C Huber, Nigel M Crawford
    Abstract:

    Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this Post-Translational Regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for Post-Translational Regulation in vitro.

Lalita A Shevde - One of the best experts on this subject based on the ideXlab platform.

  • loss of tumor suppressor merlin in advanced breast cancer is due to post translational Regulation
    Journal of Biological Chemistry, 2011
    Co-Authors: Adam K Morrow, Shamik Das, Brandon J Metge, Madhuri S Mulekar, Allan J Tucker, Rajeev S Samant, Lalita A Shevde
    Abstract:

    Unlike malignancies of the nervous system, there have been no mutations identified in Merlin in breast cancer. As such, the role of the tumor suppressor, Merlin, has not been investigated in breast cancer. We assessed Merlin expression in breast cancer tissues by immunohistochemistry and by real-time PCR. The expression of Merlin protein (assessed immunohistochemically) was significantly decreased in breast cancer tissues (although the transcript levels were comparable) simultaneous with increased expression of the tumor-promoting protein, osteopontin (OPN). We further demonstrate that the loss of Merlin in breast cancer is brought about, in part, due to OPN-initiated Akt-mediated phosphorylation of Merlin leading to its proteasomal degradation. Restoring expression of Merlin resulted in reduced malignant attributes of breast cancer, characterized by reduced invasion, migration, motility, and impeded tumor (xenograft) growth in immunocompromised mice. The possibility of developing a model using the relationship between OPN and Merlin was tested with a logistic regression model applied to immunohistochemistry data. This identified consistent loss of immunohistochemical expression of Merlin in breast tumor tissues. Thus, we demonstrate for the first time a role for Merlin in impeding breast malignancy, identify a novel mechanism for the loss of Merlin protein in breast cancer, and have developed a discriminatory model using Merlin and OPN expression in breast tumor tissues.

Daniel J Klionsky - One of the best experts on this subject based on the ideXlab platform.

Lynne P Howell - One of the best experts on this subject based on the ideXlab platform.

  • dimeric c di gmp is required for post translational Regulation of alginate production in pseudomonas aeruginosa
    Journal of Biological Chemistry, 2015
    Co-Authors: John C Whitney, Howard Robinson, Gregory B Whitfield, Lindsey S Marmont, Mirela A Neculai, Y D Lobsanov, Dennis E Ohman, Lynne P Howell
    Abstract:

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity Post-Translationally regulates alginate secretion. In this study, we report the 1.8 A crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor producedmore » by P. aeruginosa.« less