scholarly journals Escherichia coli SecG Is Required for Residual Export Mediated by Mutant Signal Sequences and for SecY-SecE Complex Stability

2014 ◽  
Vol 197 (3) ◽  
pp. 542-552 ◽  
Author(s):  
Dominique Belin ◽  
Giuseppe Plaia ◽  
Yasmine Boulfekhar ◽  
Filo Silva
Keyword(s):  
Critical Role ◽  
Standard Laboratory ◽  
Loss Of Function ◽  
Wild Type ◽  
Limited Effect ◽  
Signal Sequences ◽  
Biochemical Basis ◽  
Mutant Strains ◽  
The Stability ◽  
Synthetic Phenotype

Protein export to the bacterial periplasm is achieved by SecYEG, an inner membrane heterotrimer. SecY and SecE are encoded by essential genes, while SecG is not essential for growth under standard laboratory conditions. Using a quantitative and sensitive export assay, we show that SecG plays a critical role for the residual export mediated by mutant signal sequences; the magnitude of this effect is not proportional to the strength of the export defect. In contrast, export mediated by wild-type signal sequences is only barely retarded in the absence of SecG. When probed with mutant signal sequences,secGloss of function mutations display a phenotype opposite to that ofprlAmutations insecY. The analysis ofsecGandprlAsingle and double mutant strains shows that the increased export conferred by severalprlAalleles is enhanced in the absence of SecG. Several combinations ofprlAalleles with asecGdeletion cannot be easily constructed. This synthetic phenotype is conditional, indicating that cells can adapt to the presence of both alleles. The biochemical basis of this phenomenon is linked to the stability of the SecYE dimer in solubilized membranes. WithprlAalleles that can be normally introduced in asecGdeletion strain, SecG has only a limited effect on the stability of the SecYE dimer. With the otherprlAalleles, the SecYE dimer can often be detected only in the presence of SecG. A possible role for the maintenance of SecG during evolution is proposed.

scholarly journals act Operon Control of Developmental Gene Expression in Myxococcus xanthus

2002 ◽  
Vol 184 (4) ◽  
pp. 1172-1179 ◽  
Author(s):  
Thomas M. A. Gronewold ◽  
Dale Kaiser
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
The Other ◽  
Loss Of Function ◽  
Wild Type ◽  
Developmental Gene ◽  
Developmental Gene Expression ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Signal Production

ABSTRACT Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.

scholarly journals Adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality

2008 ◽  
Vol 295 (3) ◽  
pp. E658-E664 ◽  
Author(s):  
Hwee Teoh ◽  
Adrian Quan ◽  
K. W. Annie Bang ◽  
Guilin Wang ◽  
Fina Lovren ◽  
...  
Keyword(s):  
Adhesion Molecule ◽  
Critical Role ◽  
Cecal Ligation ◽  
Endothelial Activation ◽  
Experimental Sepsis ◽  
Protective Effects ◽  
Loss Of Function ◽  
Wild Type ◽  
Endothelial Adhesion Molecule ◽  
Inflammatory Models

Sepsis is a multifactorial, and often fatal, disorder typically characterized by widespread inflammation and immune activation with resultant endothelial activation. In the present study, we postulated that the adipokine adiponectin serves as a critical modulator of survival and endothelial activation in sepsis. To this aim, we evaluated both loss-of-function (adiponectin gene-deficient mice) and subsequent gain-of-function (recombinant adiponectin reconstitution) strategies in two well-established inflammatory models, cecal ligation perforation (CLP) and thioglyocollate-induced peritonitis. Adipoq−/− mice, subjected to CLP, exhibited a profound (∼8-fold) reduction in survival compared with their wild-type Adipoq+/+ littermates after 48 h. Furthermore, compared with wild-type controls, thioglycollate challenge resulted in a markedly greater influx of peritoneal neutrophils in Adipoq−/− mice accompanied by an excess production of key chemoattractant cytokines (IL-12p70, TNFα, MCP-1, and IL-6) and upregulation of aortic endothelial adhesion molecule VCAM-1 and ICAM-1 expressions. Importantly, all of these effects were blunted by recombinant total adiponectin administration given 3 days prior to thioglycollate challenge. The protective effects of adiponectin were ascribed largely to higher-order adiponectin oligomers, since administration of recombinant C39A trimeric adiponectin did not attenuate endothelial adhesion molecule expression in thioglycollate-challenged Adipoq−/− mice. These data suggest a critical role of adiponectin as a modulator of survival and endothelial inflammation in experimental sepsis and a potential mechanistic link between adiposity and increased sepsis.

An adenylyl cyclase that functions during late development of Dictyostelium

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5463-5471 ◽  
Author(s):  
F. Soderbom ◽  
C. Anjard ◽  
N. Iranfar ◽  
D. Fuller ◽  
W.F. Loomis
Keyword(s):  
Adenylyl Cyclase ◽  
Critical Role ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Adenylyl Cyclase Activity ◽  
Extracellular Signals ◽  
Mutant Strains ◽  
Morphological Mutants ◽  
Low Levels ◽  
Mutant Cells

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(−) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(−) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(−) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.

scholarly journals Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

2010 ◽  
Vol 84 (16) ◽  
pp. 8072-8084 ◽  
Author(s):  
Sarah E. Hobdey ◽  
Brian J. Kempf ◽  
Benjamin P. Steil ◽  
David J. Barton ◽  
Olve B. Peersen
Keyword(s):  
Amino Acid ◽  
Rna Binding ◽  
Critical Role ◽  
Polymerase Activity ◽  
Structural Basis ◽  
Wild Type ◽  
Elongation Complex ◽  
Virus Growth ◽  
The Stability

ABSTRACT The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.

scholarly journals EPEN-20. EZHIP/CATACOMB COOPERATES WITH PDGF-A AND p53 LOSS TO GENERATE A GENETICALLY ENGINEERED MOUSE MODEL FOR POSTERIOR FOSSA A EPENDYMOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii311-iii311
Author(s):  
Emily Kagan ◽  
Daniel Brat ◽  
Ali Shilatifard ◽  
Andrea Piunti ◽  
Oren Becher
Keyword(s):  
Median Survival ◽  
Critical Role ◽  
Pediatric Brain Tumor ◽  
Genetically Engineered ◽  
Rank Test ◽  
Loss Of Function ◽  
Wild Type ◽  
Term Survival ◽  
Log Rank Test ◽  
Genetically Engineered Mouse Model

Abstract BACKGROUND PFA ependymoma is a pediatric brain tumor with only 30% long-term survival. Recently a gene called CXORF67/EZHIP/CATACOMB (henceforward: CATACOMB) was found to be overexpressed in PFA ependymoma. CATACOMB’s mechanism of action has been found to be analogous to that of the H3K27M mutation as its expression reduces H3K27me3 via inhibition of PRC2 catalytic activity. METHODS We infected NESTIN- or GFAP-expressing neonatal hindbrain progenitors with wild-type CATACOMB or a loss of function (LOF) point mutant (M406K), alone, with PDGFA, and with and without p53 deletion. RESULTS CATACOMB overexpression alone or with p53 loss was insufficient to induce tumorigenesis. CATACOMB overexpression with PDGFA and p53 loss was sufficient to induce tumorigenesis using either the LOF mutant (M406K) or the wild-type CATACOMB in both cells-of-origin. The histology appeared more ependymoma-like when CATACOMB was expressed in GFAP-expressing progenitors. Median survival for the model initiated in NESTIN progenitors was 99.5 days for the CATACOMB mutant (n=26) group and 61 days for the CATACOMB wild-type (n=28; log-rank test p=0.0033). Median survival for the model initiated in GFAP progenitors were 144 days for the CATACOMB mutant (n=19) group and 65 days for the CATACOMB wild-type (n=21; log-rank test is P<0.0013). Immunohistochemistry for H3K27me3 demonstrated that CATACOMB wild-type tumors had reduced H3K27me3 compared to CATACOMB mutant tumors. CONCLUSIONS Disrupting CATACOMB inhibitory activity toward PRC2 significantly increases survival in mice in both models, suggesting this activity plays a critical role in accelerating tumorigenesis. Ependymoma-like histology was more commonly observed in the model initiated in the GFAP-expressing progenitors.

scholarly journals Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity

2021 ◽  
Author(s):  
Yang-Yang Li ◽  
Rong-Jun Cai ◽  
Jia-Ying Yang ◽  
Tamara L. Hendrickson ◽  
Ye Xiang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Second Step ◽  
Loss Of Function ◽  
Wild Type ◽  
Translational Fidelity ◽  
Partial Loss ◽  
Biophysical Characterization ◽  
Minimal Impact ◽  
Mutant Strains

AbstractMost bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error-prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampicin tolerance. However, the mechanisms by which these clinically-derived mutations in gatA impact GatCAB function was unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains, but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.

scholarly journals BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast.

1995 ◽  
Vol 130 (1) ◽  
pp. 41-49 ◽  
Author(s):  
J F Simons ◽  
S Ferro-Novick ◽  
M D Rose ◽  
A Helenius
Keyword(s):  
Molecular Chaperone ◽  
Intracellular Transport ◽  
Critical Role ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Yeast Strains ◽  
Mutant Strains

Although transiently associated with numerous newly synthesized proteins, BiP has not been shown to be an essential component directly linked to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a molecular chaperone, we analyzed the maturation of an endogenous yeast glycoprotein, carboxypeptidase Y (CPY) in several yeast strains with temperature-sensitive mutations in BiP. These kar2 mutant strains have previously been found to be defective in translocation at the nonpermissive temperature (Vogel, J. P., L. M. Misra, and M. D. Rose, 1990. J. Cell Biol, 110:1885-1895). To circumvent the translocation block, we used DTT at permissive temperature to delay folding and intracellular transport. We then followed the maturation of the ER-retained CPY after shifting to the nonpermissive temperature and dilution of the DTT. Without the functional chaperone, CPY aggregated, failed to be oxidized, and remained in the ER. In contrast to wild-type cells, in which BiP binding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the mutant strains. In a heterozygous diploid strain, CPY matured and exited the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding.

MicroRNA-21-5p as a novel therapeutic target for osteoarthritis

Rheumatology ◽  
2019 ◽  
Vol 58 (8) ◽  
pp. 1485-1497 ◽  
Author(s):  
Xiao-bo Wang ◽  
Feng-chao Zhao ◽  
Lin-hong Yi ◽  
Jin-long Tang ◽  
Zheng-ya Zhu ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Medial Meniscus ◽  
Critical Role ◽  
Cartilage Degradation ◽  
Mirna Expression Profile ◽  
Conditional Knockout ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Wild Type ◽  
Functional Pathway

Abstract Objective Growing evidence indicates that microRNAs (miRNA) play a critical role in the pathogenesis of OA, and overexpressing or silencing miRNA expression in OA models can contribute to the development of miRNA-based therapeutics. The objective of this study was to determine whether intra-articular injection of miRNA can inhibit OA progression. Methods The miRNA expression profile was determined in OA cartilage tissues and controls. Functional analysis of the miRNAs on extracellular matrix degradation was performed after miRNA mimic or inhibitor transfection. Luciferase reporter assays and western blotting were employed to determine miRNA targets. To investigate the functional mechanism of miR-21-5p in OA development, miR-21-5pfl/flCol2a1-CreER and wild-type mice were subject to surgical destabilization of the medial meniscus. Therapeutically, wild-type mice undergoing surgical destabilization of the medial meniscus were treated with intra-articular injection of agomir- and antagomir-21-5p. Results We found that expression of miR-21-5p was significantly up-regulated in OA cartilage tissues. The articular cartilage degradation of miR-21-5p conditional knockout mice was significantly alleviated compared with that of wild-type mice in spontaneous and destabilization of the medial meniscus models. Through gain-of-function and loss-of-function studies, miR-21-5p was shown to significantly affect matrix synthesis genes expression, and chondrocyte proliferation and apoptosis. Further, fibroblast growth factor 18 (FGF18) was identified as a target of miR-21-5p. Intra-articular injection of antagomir-21-5p significantly attenuated the severity of experimental OA. Clinically, FGF18 expression level was correlated with miR-21-5p expression and a modified Mankin scale. Conclusion Our findings reveal a miRNA functional pathway important for OA development, highlighting miRNA-21-5p silencing as an attractive therapeutic regimen in future clinical trials involving patients with OA.

scholarly journals Effects of a Temperature Sensitivity Mutation in the J1R Protein Component of a Complex Required for Vaccinia Virus Assembly

2005 ◽  
Vol 79 (13) ◽  
pp. 8046-8056 ◽  
Author(s):  
Wen-Ling Chiu ◽  
Patricia Szajner ◽  
Bernard Moss ◽  
Wen Chang
Keyword(s):  
Vaccinia Virus ◽  
Virus Assembly ◽  
Terminal Region ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Wild Type ◽  
Reading Frame ◽  
Infected Cells ◽  
The Stability ◽  
Virus C

ABSTRACT Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39°C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39°C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.

scholarly journals The SAM Domains of Anks Family Proteins Are Critically Involved in Modulating the Degradation of EphA Receptors

2010 ◽  
Vol 30 (7) ◽  
pp. 1582-1592 ◽  
Author(s):  
Jieun Kim ◽  
Haeryung Lee ◽  
Yujin Kim ◽  
Sooyeon Yoo ◽  
Eunjeong Park ◽  
...  
Keyword(s):  
Potential Role ◽  
Critical Role ◽  
Dominant Negative ◽  
Eph Receptors ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Sam Domain ◽  
Ligand Stimulation ◽  
The Stability

ABSTRACT We recently reported that the phosphotyrosine-binding (PTB) domain of Anks family proteins binds to EphA8, thereby positively regulating EphA8-mediated signaling pathways. In the current study, we identified a potential role for the SAM domains of Anks family proteins in EphA signaling. We found that SAM domains of Anks family proteins directly bind to ubiquitin, suggesting that Anks proteins regulate the degradation of ubiquitinated EphA receptors. Consistent with the role of Cbl ubiquitin ligases in the degradation of Eph receptors, our results revealed that the ubiquitin ligase c-Cbl induced the ubiquitination and degradation of EphA8 upon ligand binding. Ubiquitinated EphA8 also bound to the SAM domains of Odin, a member of the Anks family proteins. More importantly, the overexpression of wild-type Odin protected EphA8 and EphA2 from undergoing degradation following ligand stimulation and promoted EphA-mediated inhibition of cell migration. In contrast, a SAM domain deletion mutant of Odin strongly impaired the function of endogenous Odin, suggesting that the mutant functions in a dominant-negative manner. An analysis of Odin-deficient primary embryonic fibroblasts indicated that Odin levels play a critical role in regulating the stability of EphA2 in response to ligand stimulation. Taken together, our studies suggest that the SAM domains of Anks family proteins play a pivotal role in enhancing the stability of EphA receptors by modulating the ubiquitination process.

Sign in / Sign up

Export Citation Format

Share Document