scholarly journals Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

2009 ◽  
Vol 191 (22) ◽  
pp. 7086-7093 ◽  
Author(s):  
Cheryl P. Ewing ◽  
Ekaterina Andreishcheva ◽  
Patricia Guerry
Keyword(s):  
Campylobacter Jejuni ◽  
Functional Characterization ◽  
Subunit Interactions ◽  
Independent Manner ◽  
Structural Subunit ◽  
Flagellar Filament ◽  
Flagellar Regulon ◽  
Multiple Levels ◽  
Flagellin Glycosylation

ABSTRACT The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.

scholarly journals Identification of diverse targets of arginine phosphorylation in Mycolicibacterium smegmatis by shotgun proteomics

2021 ◽  
Author(s):  
Emmanuel C Ogbonna ◽  
Karl R Schmitz
Keyword(s):  
Quality Control ◽  
Drug Targets ◽  
Functional Characterization ◽  
Shotgun Proteomics ◽  
Multidrug Resistant ◽  
Phosphopeptide Enrichment ◽  
Independent Manner ◽  
New Evidence ◽  
Lines Of Evidence

Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. Several lines of evidence suggest that ClpC1P1P2 similarly recognizes phosphoarginine-bearing proteins, but the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate of Mtb. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on a diverse collection of targets within the Msm proteome. Physicochemical and functional characterization of targets suggest that arginine phosphorylation is applied in a sequence-independent manner as part of a proteome-wide quality control pathway. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species.

scholarly journals The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression

2021 ◽  
Vol 14 (2) ◽  
pp. dmm046300
Author(s):  
Dan Wu ◽  
Sailan Wang ◽  
Daniel V. Oliveira ◽  
Francesca Del Gaudio ◽  
Michael Vanlandewijck ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Extracellular Domain ◽  
Infantile Myofibromatosis ◽  
Independent Manner ◽  
Functional Link ◽  
Downstream Signaling ◽  
Chloroquine Treatment ◽  
Notch3 Mutation ◽  
The Relationship

ABSTRACTInfantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal-dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this study, we address the molecular consequences of the NOTCH3L1519P mutation and the relationship between Notch and PDGFRB signaling in IMF. The NOTCH3L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3L1519P extracellular domain and decreases signaling. Finally, NOTCH3L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3–PDGFRB axis in IMF, in which an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.

scholarly journals Functional Characterization of a Lipoprotein-Encoding Operon in Campylobacter jejuni

PLoS ONE ◽  
2011 ◽  
Vol 6 (5) ◽  
pp. e20084 ◽  
Author(s):  
Mayumi Oakland ◽  
Byeonghwa Jeon ◽  
Orhan Sahin ◽  
Zhangqi Shen ◽  
Qijing Zhang
Keyword(s):  
Campylobacter Jejuni ◽  
Functional Characterization

Characterization of new DsbB-like thiol-oxidoreductases of Campylobacter jejuni and Helicobacter pylori and classification of the DsbB family based on phylogenomic, structural and functional criteria

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 219-231 ◽  
Author(s):  
Anna M. Raczko ◽  
Janusz M. Bujnicki ◽  
Marcin Pawłowski ◽  
Renata Godlewska ◽  
Magdalena Lewandowska ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Campylobacter Jejuni ◽  
Functional Characterization ◽  
Transmembrane Helix ◽  
Bacterial Cells ◽  
Terminal Domain ◽  
Family Based ◽  
Comprehensive Classification

In Gram-negative bacterial cells, disulfide bond formation occurs in the oxidative environment of the periplasm and is catalysed by Dsb (disulfide bond) proteins found in the periplasm and in the inner membrane. In this report the identification of a new subfamily of disulfide oxidoreductases encoded by a gene denoted dsbI, and functional characterization of DsbI proteins from Campylobacter jejuni and Helicobacter pylori, as well as DsbB from C. jejuni, are described. The N-terminal domain of DsbI is related to DsbB proteins and comprises five predicted transmembrane segments, while the C-terminal domain is predicted to locate to the periplasm and to fold into a β-propeller structure. The dsbI gene is co-transcribed with a small ORF designated dba ( dsbI-accessory). Based on a series of deletion and complementation experiments it is proposed that DsbB can complement the lack of DsbI but not the converse. In the presence of DsbB, the activity of DsbI was undetectable, hence it probably acts only on a subset of possible substrates of DsbB. To reconstruct the principal events in the evolution of DsbB and DsbI proteins, sequences of all their homologues identifiable in databases were analysed. In the course of this study, previously undetected variations on the common thiol-oxidoreductase theme were identified, such as development of an additional transmembrane helix and loss or migration of the second pair of Cys residues between two distinct periplasmic loops. In conjunction with the experimental characterization of two members of the DsbI lineage, this analysis has resulted in the first comprehensive classification of the DsbB/DsbI family based on structural, functional and evolutionary criteria.

scholarly journals Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori

2003 ◽  
Vol 48 (6) ◽  
pp. 1579-1592 ◽  
Author(s):  
M. Schirm ◽  
E. C. Soo ◽  
A. J. Aubry ◽  
J. Austin ◽  
P. Thibault ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Functional Characterization ◽  
Flagellin Glycosylation

Particle characterization of CVD tungsten metallization for 64 MBIT DRAM

1991 ◽  
Vol 49 ◽  
pp. 896-897
Author(s):  
Marylyn Bennett-Lilley ◽  
Thomas T.H. Fu ◽  
David D. Yin ◽  
R. Allen Bowling
Keyword(s):  
Chemical Vapor ◽  
Device Performance ◽  
Particle Characterization ◽  
Particle Generation ◽  
Tungsten Hexafluoride ◽  
Homogeneous Particle ◽  
Multiple Levels ◽  
Circuit Density ◽  
Sources Of Contamination

Chemical Vapor Deposition (CVD) tungsten metallization is used to increase VLSI device performance due to its low resistivity, and improved reliability over other metallization schemes. Because of its conformal nature as a blanket film, CVD-W has been adapted to multiple levels of metal which increases circuit density. It has been used to fabricate 16 MBIT DRAM technology in a manufacturing environment, and is the metallization for 64 MBIT DRAM technology currently under development. In this work, we investigate some sources of contamination. One possible source of contamination is impurities in the feed tungsten hexafluoride (WF6) gas. Another is particle generation from the various reactor components. Another generation source is homogeneous particle generation of particles from the WF6 gas itself. The purpose of this work is to investigate and analyze CVD-W process-generated particles, and establish a particle characterization methodology.

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