scholarly journals Mutational Analysis of Vpr-Induced G2Arrest, Nuclear Localization, and Cell Death in Fission Yeast

1999 ◽  
Vol 73 (4) ◽  
pp. 3236-3245 ◽  
Author(s):  
Mingzhong Chen ◽  
Robert T. Elder ◽  
Min Yu ◽  
Maurice G. O’Gorman ◽  
Luc Selig ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Fission Yeast ◽  
Nuclear Localization ◽  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Cell Killing ◽  
Human Cells ◽  
Α Helix

ABSTRACT Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal α-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each α-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.

scholarly journals Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
Hairul Aini Hamzah ◽  
Rahmatullah Sirat ◽  
Mohammed Imad A. Mustafa Mahmud ◽  
Roesnita Baharudin
Keyword(s):  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Resistant Bacteria ◽  
Single Point Mutation ◽  
Phenotypic Resistance ◽  
Drug Effectiveness ◽  
E Coli

 Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

scholarly journals Recombinant Helicobacter pylori CagA protein induces endoplasmic reticulum stress and autophagy in human cells

2020 ◽  
Author(s):  
Babak Nami ◽  
Ali Azzawri ◽  
Vasfiye B Ucar ◽  
Hasan Acar
Keyword(s):  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Helicobacter Pylori ◽  
Western Blot ◽  
Er Stress ◽  
Nuclear Localization ◽  
Human Cell ◽  
Human Cells ◽  
Caga Protein

AbstractHelicobacter pylori (Hp) CagA protein has a key role in the development of gastric cancer by the intruding in many intracellular processes of host human cell. Endoplasmic reticulum (ER) stress is an essential process for cellular homeostasis that modulates survival and death and is linked to several complex diseases including cancer. CagA protein is found in the serum of Hp-positive individuals and also in the supernatant of Hp culture. Limited studies report that recombinant CagA can alter gene expression and signaling pathways and induce the death of human cells. In this study, we investigated the effect of exogenous recombinant CagA protein treatment on ER stress and autophagy of human cell. AGS, MKN45, and HEK293 cells were treated with 1 µg/ml of recombinant CagA protein and then ER stress was studied by quantitative-PCR of spliced XBP-1 mRNA, immunofluorescence staining of ATF6 protein nuclear localization and real-time quantitative-PCR and/or western blot expression of GRP78, GRP94, ATF4 and CHOP genes. Autophagy was studied by western blot assessment of the conversion of LC3-I to LC3-II and LC3 aggregation. Cell proliferation and death were investigated by MTT assay and trypan blue staining respectively. As result, treatment with recombinant CagA enhanced XBP-1mRNA splicing, nuclear localization of ATF6, and the expression of ER stress signaling target genes in the cells. Recombinant CagA also induced LC3 protein conversion and aggregation in the cells. Reduced cell proliferation and increased cell death were determined in the cells treated with recombinant CagA. These results show that exogenous recombinant CagA protein causes cell death by inducing ER stress and autophagy in human cells. We conclude that CagA protein exogenously localizes in/on human cells and induces ER stress via disturbing protein machinery leading the human cell to death, however, the mechanism of CagA-host cell interaction is to be investigated.

scholarly journals A Role for the START Gene–specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

2000 ◽  
Vol 11 (10) ◽  
pp. 3411-3424 ◽  
Author(s):  
Sylvie Tournier ◽  
Jonathan B.A. Millar
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Fission Yeast ◽  
Single Point ◽  
Cyclin B ◽  
Mitotic Catastrophe ◽  
Single Point Mutation ◽  
Restrictive Temperature ◽  
Specific Transcription Factor ◽  
Transcription Factor Complex

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature. Eleven of 17 mutants that suppress this phenotype define a single complementation group, mcs1. The mcs1-77mutant also suppresses lethal inactivation of the Wee1 and Mik1 tyrosine kinases and thus delays mitosis independently of Cdc2 tyrosine phosphorylation. We have cloned mcs1 by isolating suppressors of the cell cycle arrest phenotype of mcs1-77 cdc25-22 cells and found that it encodes Res2, a component of the START gene–specific transcription factor complex MBF (also known as DSC-1). The mcs1-77 mutant bears a single point mutation in the DNA-binding domain of Res2 that causes glycine 68 to be replaced by a serine residue. Importantly, two substrates of the anaphase-promoting complex (APC), the major B-type cyclin, Cdc13, and the anaphase inhibitor, Cut2, are unstable in G2-phasemcs1-77 cells. Consistent with this, we observe abnormal sister chromatid separation in mcs1-77 cdc25-22 cells at the restrictive temperature. Mutation of either Cdc10 or Res1 also deregulates MBF-dependent transcription and causes a G2 delay. We find that this cell cycle delay is abolished in the absence of the APC regulator Ste9/Srw1 and that the periodic expression of Ste9/Srw1 is controlled by the MBF complex. These data suggest that in fission yeast the MBF complex plays a key role in the inactivation of cyclin B and Cut2 destruction by controlling the periodic production of APC regulators.

scholarly journals Characterization of cytopathic factors through genome-wide analysis of the Zika viral proteins in fission yeast

2017 ◽  
Vol 114 (3) ◽  
pp. E376-E385 ◽  
Author(s):  
Ge Li ◽  
Melissa Poulsen ◽  
Csaba Fenyvuesvolgyi ◽  
Yoko Yashiroda ◽  
Minoru Yoshida ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Fission Yeast ◽  
Nonstructural Protein ◽  
Intracellular Localization ◽  
Protein S ◽  
Growth Restriction ◽  
Mechanistic Study ◽  
Future Research ◽  
Cytopathic Effects

The Zika virus (ZIKV) causes microcephaly and the Guillain-Barré syndrome. Little is known about how ZIKV causes these conditions or which ZIKV viral protein(s) is responsible for the associated ZIKV-induced cytopathic effects, including cell hypertrophy, growth restriction, cell-cycle dysregulation, and cell death. We used fission yeast for the rapid, global functional analysis of the ZIKV genome. All 14 proteins or small peptides were produced under an inducible promoter, and we measured the intracellular localization and the specific effects on ZIKV-associated cytopathic activities of each protein. The subcellular localization of each ZIKV protein was in overall agreement with its predicted protein structure. Five structural and two nonstructural ZIKV proteins showed various levels of cytopathic effects. The expression of these ZIKV proteins restricted cell proliferation, induced hypertrophy, or triggered cellular oxidative stress leading to cell death. The expression of premembrane protein (prM) resulted in cell-cycle G1 accumulation, whereas membrane-anchored capsid (anaC), membrane protein (M), envelope protein (E), and nonstructural protein 4A (NS4A) caused cell-cycle G2/M accumulation. A mechanistic study revealed that NS4A-induced cellular hypertrophy and growth restriction were mediated specifically through the target of rapamycin (TOR) cellular stress pathway involving Tor1 and type 2A phosphatase activator Tip41. These findings should provide a reference for future research on the prevention and treatment of ZIKV diseases.

scholarly journals Identification of a Turnover Element in Region 2.1 of Escherichia coli σ32 by a Bacterial One-Hybrid Approach

2005 ◽  
Vol 187 (11) ◽  
pp. 3807-3813 ◽  
Author(s):  
Markus Obrist ◽  
Franz Narberhaus
Keyword(s):  
Escherichia Coli ◽  
Sigma Factor ◽  
Transcriptional Activity ◽  
Single Point ◽  
Hybrid Approach ◽  
Point Mutations ◽  
Alternative Sigma Factor ◽  
Single Point Mutations ◽  
Α Helix ◽  
Structure Calculations

ABSTRACT Induction of the heat shock response in Escherichia coli requires the alternative sigma factor σ32 (RpoH). The cellular concentration of σ32 is controlled by proteolysis involving FtsH, other proteases, and the DnaKJ chaperone system. To identify individual σ32 residues critical for degradation, we used a recently developed bacterial one-hybrid system and screened for stabilized versions of σ32. The five single point mutations that rendered the sigma factor more stable mapped to positions L47, A50, and I54 in region 2.1. Strains expressing the stabilized σ32 variants exhibited elevated transcriptional activity, as determined by a groE-lacZ fusion. Structure calculations predicted that the three mutated residues line up on the same face of an α-helix in region 2.1, suggesting that they are positioned to interact with proteins of the degradation machinery.

scholarly journals Identification of Essential Residues in the Erm(B) rRNA Methyltransferase of Clostridium perfringens

2002 ◽  
Vol 46 (5) ◽  
pp. 1253-1261 ◽  
Author(s):  
Kylie A. Farrow ◽  
Dena Lyras ◽  
Galina Polekhina ◽  
Katerina Koutsis ◽  
Michael W. Parker ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Mutational Analysis ◽  
Single Point ◽  
Protein A ◽  
Point Mutations ◽  
23S Rrna ◽  
Mutator Strain ◽  
Rational Development ◽  
Hydroxylamine Mutagenesis

ABSTRACT Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.

scholarly journals Induction of p53-Independent Apoptosis by the Adenovirus E4orf4 Protein Requires Binding to the Bα Subunit of Protein Phosphatase 2A

2000 ◽  
Vol 74 (17) ◽  
pp. 7869-7877 ◽  
Author(s):  
Richard C. Marcellus ◽  
Helen Chan ◽  
Denis Paquette ◽  
Sarah Thirlwell ◽  
Dominique Boivin ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Point Mutations ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Cell Killing ◽  
Productive Infection ◽  
Viral Spread ◽  
Phosphatase 2A

ABSTRACT Previous studies have indicated that the E4orf4 protein of human adenovirus type 2 (Ad2) induces p53-independent apoptosis. We believe that this process may play a role in cell death and viral spread at the final stages of productive infection. E4orf4 may also be of therapeutic value in treating some diseases, including cancer, through its ability to induce apoptosis when expressed individually. The only previously identified biochemical function of E4orf4 is its ability to associate with the Bα subunit of protein phosphatase 2A (PP2A). We have used a genetic approach to determine the role of such interactions in E4orf4-induced cell death. E4orf4 deletion mutants were of only limited value, as all were highly defective. We found that E4orf4 proteins from most if not all adenovirus serotypes induced cell death, and thus point mutations were introduced that converted the majority of highly conserved residues to alanines. Such mutants were used to correlate Bα-subunit binding, association with PP2A activity, and cell killing following the transfection of appropriate cDNAs into p53-null H1299 or C33A cells. The results indicated that binding of the Bα subunit is essential for induction of cell death, as every mutant that failed to bind efficiently was totally defective for cell killing. This class of mutations (class I) largely involved residues between amino acids 51 and 89. Almost all E4orf4 mutant proteins that associated with PP2A killed cancer cells at high levels; however, several mutants that associated with significant levels of PP2A were defective for killing (class II). Thus, binding of E4orf4 to PP2A is essential for induction of p53-independent apoptosis, but E4orf4 may possess one or more additional functions required for cell killing.

scholarly journals Mutational Analysis of the Rous Sarcoma Virus DR Posttranscriptional Control Element

1998 ◽  
Vol 72 (4) ◽  
pp. 3407-3411 ◽  
Author(s):  
Robert A. Ogert ◽  
Karen L. Beemon
Keyword(s):  
Rous Sarcoma Virus ◽  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Viral Rna ◽  
Control Element ◽  
Posttranscriptional Control ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking src and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.

Sign in / Sign up

Export Citation Format

Share Document