scholarly journals Identification of the Helicobacter pylori VacA Toxin Domain Active in the Cell Cytosol

1998 ◽  
Vol 66 (12) ◽  
pp. 6014-6016 ◽  
Author(s):  
Marina de Bernard ◽  
Daniela Burroni ◽  
Emanuele Papini ◽  
Rino Rappuoli ◽  
John Telford ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Hela Cells ◽  
Terminal Region ◽  
Endocytic Pathway ◽  
Amino Terminal ◽  
Toxin Molecule ◽  
Intracellular Activity ◽  
Cell Cytosol ◽  
C And N ◽  
Late Stages

ABSTRACT Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity.

scholarly journals SEL-5, A Serine/Threonine Kinase That Facilitates lin-12 Activity in Caenorhabditis elegans

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1641-1654 ◽  
Author(s):  
Hanna Fares ◽  
Iva Greenwald
Keyword(s):  
Cell Fate ◽  
Point Mutations ◽  
Kinase Domain ◽  
Terminal Region ◽  
Serine Threonine Kinase ◽  
Intracellular Domain ◽  
Cell Fate Decisions ◽  
Threonine Kinase ◽  
Amino Terminal ◽  
Fate Decision

Abstract Ligands present on neighboring cells activate receptors of the LIN-12/Notch family by inducing a proteolytic cleavage event that releases the intracellular domain. Mutations that appear to eliminate sel-5 activity are able to suppress constitutive activity of lin-12(d) mutations that are point mutations in the extracellular domain of LIN-12, but cannot suppress lin-12(intra), the untethered intracellular domain. These results suggest that sel-5 acts prior to or during ligand-dependent release of the intracellular domain. In addition, sel-5 suppression of lin-12(d) mutations is tissue specific: loss of sel-5 activity can suppress defects in the anchor cell/ventral uterine precursor cell fate decision and a sex myoblast/coelomocyte decision, but cannot suppress defects in two different ventral hypodermal cell fate decisions in hermaphrodites and males. sel-5 encodes at least two proteins, from alternatively spliced mRNAs, that share an amino-terminal region and differ in the carboxy-terminal region. The amino-terminal region contains the hallmarks of a serine/threonine kinase domain, which is most similar to mammalian GAK1 and yeast Pak1p.

scholarly journals Role of the amino-terminal region of the DnaA protein in opening of the duplex DNA at theoriCregion

1999 ◽  
Vol 176 (1) ◽  
pp. 163-167 ◽  
Author(s):  
Shinji Mima ◽  
Yoshihiro Yamagachi ◽  
Taemi Kondo ◽  
Tomofusa Tsuchiya ◽  
Tohru Mizushima
Keyword(s):  
Terminal Region ◽  
Duplex Dna ◽  
Dnaa Protein ◽  
Amino Terminal

scholarly journals Molecular cloning of the amino-terminal region of a rat MUC 2 mucin gene homologue. Evidence for expression in both intestine and airway.

1994 ◽  
Vol 269 (27) ◽  
pp. 17833-17840
Author(s):  
H. Ohmori ◽  
A.F. Dohrman ◽  
M. Gallup ◽  
T. Tsuda ◽  
H. Kai ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Terminal Region ◽  
Amino Terminal ◽  
Mucin Gene ◽  
Gene Homologue

scholarly journals Stability of the Human Papillomavirus Type 18 E2 Protein Is Regulated by a Proteasome Degradation Pathway through Its Amino-Terminal Transactivation Domain

2001 ◽  
Vol 75 (16) ◽  
pp. 7244-7251 ◽  
Author(s):  
Sophie Bellanger ◽  
Caroline Demeret ◽  
Sylvain Goyat ◽  
Françoise Thierry
Keyword(s):  
Human Papillomavirus ◽  
Hela Cells ◽  
Half Life ◽  
Fluorescent Protein ◽  
Growth Arrest ◽  
Transactivation Domain ◽  
Mcf7 Cells ◽  
E2 Protein ◽  
Amino Terminal

ABSTRACT The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after transfection and then rapidly disappeared, indicating that E2 was unstable in HeLa cells and could confer instability to GFP. Similar studies done with a protein lacking the transactivation domain indicated that this truncation strongly stabilizes the E2 protein. In vitro, full-length E2 or the transactivation domain alone was efficiently ubiquitinated, whereas deletion of the transactivation domain strongly decreased the ubiquitination of the E2 protein. Proteasome inhibition in cells expressing E2 increased its half-life about sevenfold, which was comparable to the half-life of the amino-terminally truncated protein. These characteristics of E2 instability were independent of the E2-mediated G1 growth arrest in HeLa cells, as they were reproduced in MCF7 cells, where E2 does not affect the cell cycle. Altogether, these experiments showed that the HPV18 E2 protein was degraded by the ubiquitin-proteasome pathway through its amino-terminal transactivation domain. Tight regulation of the stability of the HPV 18 E2 protein may be essential to avoid accumulation of a potent transcriptional repressor and antiproliferative agent during the viral vegetative cycle.

scholarly journals Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

2015 ◽  
Vol 83 (8) ◽  
pp. 3233-3242 ◽  
Author(s):  
Lena Meyer ◽  
Jeanette E. Bröms ◽  
Xijia Liu ◽  
Martin E. Rottenberg ◽  
Anders Sjöstedt
Keyword(s):  
Listeria Monocytogenes ◽  
Francisella Tularensis ◽  
Hela Cells ◽  
Cell Types ◽  
Metabolic Adaptation ◽  
Content Type ◽  
Bacterial Uptake ◽  
J774 Cells ◽  
Cell Cytosol ◽  
Successful Replication

Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. ForFrancisella tularensis, many genes of theFrancisellapathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) ofF. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpEstrains; andListeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC−/−BMDM, MyD88−/−BMDM, J774 cells, or HeLa cells, LVS, ΔpdpEand ΔiglGmutants, andL. monocytogenesreplicated efficiently in all five cell types, whereas the ΔiglAand ΔiglCmutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC−/−BMDM, and HeLa cells. In contrast to the rapid replication in other cell types,L. monocytogenesshowed no replication in MyD88−/−BMDM and LVS showed no replication in either BMDM or MyD88−/−BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartmentper seare important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

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