scholarly journals Cdc42 Is Required for Proper Growth and Development in the Fungal Pathogen Colletotrichum trifolii

2006 ◽  
Vol 5 (1) ◽  
pp. 155-166 ◽  
Author(s):  
Changbin Chen ◽  
Young-sil Ha ◽  
Ji-young Min ◽  
Stephen D. Memmott ◽  
Martin B. Dickman
Keyword(s):  
Spore Germination ◽  
Fungal Pathogen ◽  
Hyphal Growth ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Ras Signaling ◽  
Wild Type ◽  
Colletotrichum Trifolii ◽  
Downstream Effector ◽  
Wild Type Phenotype

ABSTRACT Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (CtCdc42), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense CtCdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly

1998 ◽  
Vol 72 (10) ◽  
pp. 7885-7894 ◽  
Author(s):  
Françoise Fischer ◽  
Carola F. Stegen ◽  
Paul S. Masters ◽  
William A. Samsonoff
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
E Gene ◽  
E Protein ◽  
Wild Type Phenotype ◽  
Necessary And Sufficient ◽  
Targeted Recombination

ABSTRACT Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

scholarly journals Colletotrichum trifolii TB3 Kinase, a COT1 Homolog, Is Light Inducible and Becomes Localized in the Nucleus during Hyphal Elongation

2002 ◽  
Vol 1 (4) ◽  
pp. 626-633 ◽  
Author(s):  
Changbin Chen ◽  
Martin B. Dickman
Keyword(s):  
Fungal Pathogen ◽  
Growth And Development ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Amino Terminus ◽  
Kinase Gene ◽  
Fusion Construct ◽  
Colletotrichum Trifolii ◽  
Functional Homolog ◽  
Protein Kinase Gene

ABSTRACT Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease of alfalfa. Previously, a serine/threonine protein kinase gene from this fungus (TB3), which is a functional homolog of the Neurospora crassa COT1 kinase, has been isolated in our laboratory and appears to be associated with hyphal elongation and branching. In this report we show that light treatment rapidly induces TB3 expression and hyphal branching frequency. Western analysis showed TB3 localization in both the cytoplasm and nucleus, but not in membranes. Moreover, indirect immunofluorescence indicated that TB3 levels were most abundant in the nucleus. To further evaluate the subcellular distribution of TB3, a TB3::GFP fusion construct was inserted into C. trifolii. Results indicated that the cellular location of TB3 changed during fungal growth and development. Consistent with previous observations, TB3 was localized in both the cytoplasm and the nucleus but was preferentially localized in the nucleus during extended hyphal growth. The amino terminus of TB3 contains two relatively long polyglutamine repeats. Yeast-based assays showed that these polyglutamine tracts can activate transcription. These results suggest that TB3 may be positioned in a signaling cascade regulating proper hyphal growth and development by functioning as a transcription factor.

scholarly journals A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Qusai Al Abdallah ◽  
Tiffany S. Norton ◽  
Amy M. Hill ◽  
Lawrence L. LeClaire ◽  
Jarrod R. Fortwendel
Keyword(s):  
Aspergillus Fumigatus ◽  
Fungal Pathogen ◽  
Hyphal Growth ◽  
Invasive Disease ◽  
Ras Signaling ◽  
Specific Domain ◽  
Content Type ◽  
Ras Protein ◽  
Crucial Difference ◽  
Higher Eukaryotes

ABSTRACT Aspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in the regulation of Ras pathway activity between the human host and the fungal pathogen. Exploration of the mechanisms through which this domain regulates signaling could lead to novel antifungal therapies specifically targeting fungal Ras pathways. Ras proteins function as conserved regulators of eukaryotic growth and differentiation and are essential signaling proteins orchestrating virulence in pathogenic fungi. Here, we report the identification of a novel N-terminal domain of the RasA protein in the filamentous fungus Aspergillus fumigatus. Whereas this domain is absent in Ras homologs of higher eukaryotes, the N-terminal extension is conserved among fungi and is characterized by a short string of two to eight amino acids terminating in an invariant arginine. For this reason, we have termed the RasA N-terminal domain the invariant arginine domain (IRD). Through mutational analyses, the IRD was found to be essential for polarized morphogenesis and asexual development, with the invariant arginine residue being most essential. Although IRD truncation resulted in a nonfunctional Ras phenotype, IRD mutation was not associated with mislocalization of the RasA protein or significant changes in steady-state RasA activity levels. Mutation of the RasA IRD diminished protein kinase A (PKA) activation and resulted in decreased interaction with the Rho-type GTPase, Cdc42. Taken together, our findings reveal novel, fungus-specific mechanisms for Ras protein function and signal transduction. IMPORTANCE Aspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in the regulation of Ras pathway activity between the human host and the fungal pathogen. Exploration of the mechanisms through which this domain regulates signaling could lead to novel antifungal therapies specifically targeting fungal Ras pathways.

scholarly journals Expression of a Constitutively Active Cdc42 Homologue Promotes Development of Sclerotic Bodies but Represses Hyphal Growth in the Zoopathogenic Fungus Wangiella (Exophiala)dermatitidis

2000 ◽  
Vol 182 (17) ◽  
pp. 4941-4950 ◽  
Author(s):  
Xiangcang Ye ◽  
Paul J. Szaniszlo
Keyword(s):  
Pathogenic Fungus ◽  
Hyphal Growth ◽  
Cell Polarization ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Fungal Cell ◽  
Obvious Effect ◽  
Intracellular Signals ◽  
In The Wild ◽  
Constitutively Active

ABSTRACT In contrast to the CDC42 homologues ofSaccharomyces cerevisiae and Schizosaccharomyces pombe, the WdCDC42 gene in the human pathogenic fungus Wangiella (Exophiala)dermatitidis was found to be nonessential for cell viability. Expression of the constitutively active allelewdcdc42 G14V at 37°C induced nonpolarized growth that led to cell enlargement and multiple nucleation. The swollen cells subsequently converted into planate divided bicellular forms or multiply septated sclerotic bodies in post-log phase, when the G14V-altered protein was diminished. Thewdcdc42 G14V mutation also strongly repressed filamentous growth both in the wild-type strain and in the temperature-sensitive hyphal-form mutant Hf1. In contrast, overexpression of the dominant negative alleleswdcdc42 T19N andwdcdc42 D120A had no obvious effect on fungal-cell polarization. These results suggested that WdCdc42p plays a unique regulatory role in cellular morphogenesis in W. dermatitidis. Activation of this protein in response to extracellular or intracellular signals seems to commit its yeast-like cells to a phenotype transition that produces sclerotic bodies while repressing hyphal development.

scholarly journals Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

2021 ◽  
Author(s):  
Ashley S. Denney ◽  
Andrew D. Weems ◽  
Michael A. McMurray
Keyword(s):  
Three Dimensional ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Type Function ◽  
Negative Effect ◽  
Chaperone Interactions

ABSTRACTLife requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach to identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Chaperone inhibition liberates the mutant to enter the nucleus where it has a slight dominant-negative effect. These findings provide new insights into the effects of missense mutations.

scholarly journals Mutational analysis of the Prt1 protein subunit of yeast translation initiation factor 3.

1995 ◽  
Vol 15 (8) ◽  
pp. 4525-4535 ◽  
Author(s):  
D R Evans ◽  
C Rasmussen ◽  
P J Hanic-Joyce ◽  
G C Johnston ◽  
R A Singer ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Gene Transcript ◽  
Missense Mutations ◽  
Wild Type ◽  
Protein Subunit

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5' end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.

scholarly journals The Role of Wild-Type RAS in Oncogenic RAS Transformation

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 662
Author(s):  
Erin Sheffels ◽  
Robert L. Kortum
Keyword(s):  
Family Members ◽  
Protein Product ◽  
Therapeutic Resistance ◽  
Ras Signaling ◽  
Ras Proteins ◽  
Wild Type ◽  
Oncogenic Ras ◽  
Downstream Effector ◽  
Ras Family

The RAS family of oncogenes (HRAS, NRAS, and KRAS) are among the most frequently mutated protein families in cancers. RAS-mutated tumors were originally thought to proliferate independently of upstream signaling inputs, but we now know that non-mutated wild-type (WT) RAS proteins play an important role in modulating downstream effector signaling and driving therapeutic resistance in RAS-mutated cancers. This modulation is complex as different WT RAS family members have opposing functions. The protein product of the WT RAS allele of the same isoform as mutated RAS is often tumor-suppressive and lost during tumor progression. In contrast, RTK-dependent activation of the WT RAS proteins from the two non-mutated WT RAS family members is tumor-promoting. Further, rebound activation of RTK–WT RAS signaling underlies therapeutic resistance to targeted therapeutics in RAS-mutated cancers. The contributions of WT RAS to proliferation and transformation in RAS-mutated cancer cells places renewed interest in upstream signaling molecules, including the phosphatase/adaptor SHP2 and the RasGEFs SOS1 and SOS2, as potential therapeutic targets in RAS-mutated cancers.

scholarly journals The crosstalk between STAT3 and p53/RAS signaling controls cancer cell metastasis and cisplatin resistance via the Slug/MAPK/PI3K/AKT-mediated regulation of EMT and autophagy

Oncogenesis ◽  
2019 ◽  
Vol 8 (10) ◽  
Author(s):  
Fan Liang ◽  
Chunxia Ren ◽  
Jingshu Wang ◽  
Shuoer Wang ◽  
Lina Yang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cell ◽  
Cisplatin Resistance ◽  
Dominant Negative ◽  
Ras Signaling ◽  
Wild Type ◽  
Cell Metastasis ◽  
Wild Type P53

Abstract Chemoresistance has been the biggest obstacle in ovarian cancer treatment, and STAT3 may play an important role in chemoresistance of multiple cancers, but the underlying mechanism of STAT3 in ovarian cancer chemoresistance has long been truly illusive, particularly in association with p53 and RAS signaling. In this study, by using wild type, constitutive active, and dominant negative STAT3 constructs, wild-type p53, and RAS-mutant V12, we performed a series of in vitro and in vivo experiments by gene overexpression, drug treatment, and animal assays. We found that phosphorylation of STAT3 Y705 but not S727 promoted cancer cell EMT and metastasis through the Slug-mediated regulation of E-cadherin and Vimentin. The phosphorylation of STAT3 at Y705 also activated the MAPK and PI3K/AKT signaling to inhibit the ERS-mediated autophagy through down-regulation of pPERK, pelf2α, ATF6α, and IRE1α, which led to increased cisplatin resistance. Induction of wild type p53 in STAT3-DN-transfected cells further diminished the chemoresistance and tumor growth through the upregulation of the MAPK- and PI3K/AKT-mediated ERS and autophagy. Introduction of STAT3-DN deprived the RASV12-induced ERS, autophagy, oncogenicity, and cisplatin resistance, whereas introduction of p53 in STAT3-DN/RASV12 expressing cells induced additional tumor retardation and cisplatin sensitivity. Thus, our data provide strong evidence that the crosstalk between STAT3 and p53/RAS signaling controls ovarian cancer cell metastasis and cisplatin resistance via the Slug/MAPK/PI3K/AKT-mediated regulation of EMT and autophagy.

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