scholarly journals Further Characterization of the Signaling Proteolysis Step in the Aspergillus nidulans pH Signal Transduction Pathway

2007 ◽  
Vol 6 (6) ◽  
pp. 960-970 ◽  
Author(s):  
María M. Peñas ◽  
América Hervás-Aguilar ◽  
Tatiana Múnera-Huertas ◽  
Elena Reoyo ◽  
Miguel Á. Peñalva ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Aspergillus Nidulans ◽  
Cysteine Protease ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Western Blots ◽  
C Terminus ◽  
Catalytic Cysteine

ABSTRACT The Aspergillus nidulans pH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved “signaling protease box” in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB. In this work, we carried out classical mutational analysis of the putative signaling protease PalB, and we describe 9 missense and 18 truncating loss-of-function (including null) mutations. Mutations in the region of and affecting directly the predicted catalytic cysteine strongly support the deduction that PalB is a cysteine protease. Truncating and missense mutations affecting the C terminus highlight the importance of this region. Analysis of three-hemagglutinin-tagged PalB in Western blots demonstrates that PalB levels are independent of pH and Pal signal transduction. We have followed the processing of MYC3-tagged PacC in Western blots. We show unequivocally that PalB is essential for signaling proteolysis and is definitely not the processing protease. In addition, we have replaced 15 residues of the signaling protease box of MYC3-tagged PacC (pacC900) with alanine. The majority of these substitutions are silent. Leu481Ala, Tyr493Ala, and Gln499Ala result in delayed PacC processing in response to shifting from acidic to alkaline medium, as determined by Western blot analysis. Leu498Ala reduces function much more markedly, as determined by plate tests and processing recalcitrance. Excepting Leu498, this demonstrates that PacC signaling proteolysis is largely independent of sequence in the cleavage region.

scholarly journals Mutational Analysis of a Bifunctional Ferrisiderophore Receptor and Signal-Transducing Protein from Pseudomonas aeruginosa

2005 ◽  
Vol 187 (13) ◽  
pp. 4514-4520 ◽  
Author(s):  
H. Ellen James ◽  
Paul A. Beare ◽  
Lois W. Martin ◽  
Iain L. Lamont
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Insertion Mutagenesis ◽  
Transduction Pathway ◽  
Loss Of Function ◽  
Signaling Function ◽  
Linker Insertion Mutagenesis

ABSTRACT The FpvA protein of Pseudomonas aeruginosa strain PAO1 mediates uptake of a siderophore, ferripyoverdine. It is also a component of a signal transduction pathway that controls production of an exotoxin, a protease, pyoverdine, and FpvA itself. The purpose of the research described here was to dissect these different functions of FpvA. Signaling involves an N-terminal domain of FpvA, and it was shown that this domain is probably located in the periplasm, as expected. Short peptides were inserted at 36 sites within FpvA by linker insertion mutagenesis. The effects of these mutations on the presence of FpvA in the outer membrane, on FpvA-mediated uptake of ferripyoverdine, and on pyoverdine synthesis and gene expression were determined. Five of the mutations resulted in the absence of FpvA from the outer membrane of the bacteria. All of the remaining mutations eliminated either the transport or signaling function of FpvA and most affected both functions. Three mutations prevented transport of ferripyoverdine but had no effect on the signal transduction pathway showing that transport of ferripyoverdine is not required for the trans-membrane signaling process. Conversely, eight mutations affected pyoverdine-mediated signaling but had no effect on transport of ferripyoverdine. These data show that insertions throughout FpvA resulted in loss of function and that signaling and transport are separate and discrete functions of FpvA.

Identification of genes differentially expressed in a strain of the moldAspergillus nidulanscarrying a loss-of-function mutation in thepalAgene

2008 ◽  
Vol 54 (10) ◽  
pp. 803-811 ◽  
Author(s):  
Emiliana M. Silva ◽  
Janaína S. Freitas ◽  
Diana E. Gras ◽  
Fábio M. Squina ◽  
Juliana Leal ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Stress Responses ◽  
Genome Stability ◽  
Sugar Content ◽  
Signal Transduction Pathway ◽  
Subtractive Hybridization ◽  
Differentially Expressed ◽  
Neutral Sugar ◽  
Loss Of Function ◽  
Secretion Signal

To identify genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in palA, a gene in the pH-responsive signal transduction pathway, suppression subtractive hybridization was performed between RNA isolated from the biA1 and biA1 palA1 strains grown under limiting inorganic phosphate at pH 5.0. We have identified several genes upregulated in the biA1 palA1 mutant strain that play important roles in mitotic fidelity, stress responses, enzyme secretion, signal transduction mechanisms, development, genome stability, phosphate sensing, and transcriptional regulation among others. The upregulation of eight of these transcripts was also validated by Northern blot. Moreover, we show that a loss of function mutation in the palA gene drastically reduced the neutral sugar content of the acid phosphatase PacA secreted by the fungus A. nidulans grown at pH 5.0 compared with a control strain.

scholarly journals Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii.

1995 ◽  
Vol 131 (2) ◽  
pp. 427-440 ◽  
Author(s):  
G J Pazour ◽  
O A Sineshchekov ◽  
G B Witman
Keyword(s):  
Signal Transduction ◽  
Chlamydomonas Reinhardtii ◽  
Insertional Mutagenesis ◽  
Mutational Analysis ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Normal Manner ◽  
Normal Light ◽  
Phototransduction Pathway

Chlamydomonas has two photobehavioral responses, phototaxis and photoshock. Rhodopsin is the photoreceptor for these responses and the signal transduction process involves transmembrane Ca2+ fluxes. This causes transient changes in flagellar beating, ultimately resulting in phototaxis or photoshock. To identify components that make up this signal transduction pathway, we generated nonphototactic strains by insertional mutagenesis. Seven new phototaxis genes were identified (ptx2-ptx8); alleles of six of these are tagged by the transforming DNA and therefore should be easily cloned. To order the mutants in the pathway, we characterized them electrophysiologically, behaviorally, and structurally, ptx5, ptx6, and ptx7 have normal light-induced photoreceptor currents (PRC) and flagellar currents (FC) but their pattern of swimming does not change in the normal manner when the intraflagellar Ca2+ concentration is decreased, suggesting that they have defects in the ability of their axonemes to respond to changes in Ca2+ concentration. ptx2 and ptx8 lack the FC but have normal PRCs, suggesting that they are defective in the flagellar Ca2+ channel or some factor that regulates it. ptx4 mutants have multiple eye-spots. ptx3 mutants are defective in a component essential for phototaxis but bypassed during photoshock; this component appears to be located downstream of the PRC but upstream of the axoneme.

scholarly journals Calmodulin and Its Interactive Proteins Participate in Regulating the Explosive Growth of Alexandrium pacificum (Dinoflagellate)

2021 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
Yuan Liu ◽  
Zhimei Zhu ◽  
Zhenghong Sui ◽  
Haoxin Liu ◽  
Sadaf Riaz
Keyword(s):  
Signal Transduction ◽  
Cell Proliferation ◽  
Algal Blooms ◽  
Quantitative Polymerase Chain Reaction ◽  
Signal Transduction Pathway ◽  
Lag Phase ◽  
Transduction Pathway ◽  
Transport Channel ◽  
Western Blots ◽  
Expression Levels

Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum.

scholarly journals Isolation and Characterization of Nonchemotactic CheZ Mutants of Escherichia coli

2000 ◽  
Vol 182 (12) ◽  
pp. 3544-3552 ◽  
Author(s):  
Kristin C. Boesch ◽  
Ruth E. Silversmith ◽  
Robert B. Bourret
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Random Mutagenesis ◽  
Signal Transduction Pathway ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Functional Impairments ◽  
Biophysical Characterization ◽  
Mutant Proteins ◽  
Isolation And Characterization

ABSTRACT The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ1–189 peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.

scholarly journals Mutational Analysis of a Conserved Signal-Transducing Element: the HAMP Linker of the Escherichia coli Nitrate Sensor NarX

2003 ◽  
Vol 185 (1) ◽  
pp. 89-97 ◽  
Author(s):  
J. Alex Appleman ◽  
Valley Stewart
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Mutational Analysis ◽  
Active Role ◽  
Bound State ◽  
Missense Mutations ◽  
Structural Element ◽  
Nitrate Sensor ◽  
Domains Of Life ◽  
Sensor Proteins

ABSTRACT The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic α-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX+ strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic α-helices have different roles in signal transduction.

scholarly journals Mutational Analysis of the pH Signal Transduction Component PalC of Aspergillus nidulans Supports Distant Similarity to BRO1 Domain Family Members

Genetics ◽  
2005 ◽  
Vol 171 (1) ◽  
pp. 393-401 ◽  
Author(s):  
Joan Tilburn ◽  
Juan C. Sánchez-Ferrero ◽  
Elena Reoyo ◽  
Herbert N. Arst ◽  
Miguel A. Peñalva
Keyword(s):  
Signal Transduction ◽  
Aspergillus Nidulans ◽  
Mutational Analysis ◽  
Family Members ◽  
Domain Family

scholarly journals A Ras-mediated signal transduction pathway is involved in the control of sex myoblast migration in Caenorhabditis elegans

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2823-2833 ◽  
Author(s):  
M. Sundaram ◽  
J. Yochem ◽  
M. Han
Keyword(s):  
Signal Transduction ◽  
Caenorhabditis Elegans ◽  
Growth Factor Receptor ◽  
Signal Transduction Pathway ◽  
Body Region ◽  
Transduction Pathway ◽  
Loss Of Function ◽  
Ras Pathway ◽  
Mosaic Analysis ◽  
Sex Myoblasts

Sex myoblast migration in the Caenorhabditis elegans hermaphrodite represents a simple, genetically amenable model system for studying how cell migration is regulated during development. Two separable components of sex myoblast guidance have been described: a gonad-independent mechanism sufficient for the initial anterior migration to the mid-body region, and a gonad-dependent mechanism required for precise final positioning (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041–1052). Here, we demonstrate a role for a Ras-mediated signal transduction pathway in controlling sex myoblast migration. Loss-of-function mutations in let-60 ras, ksr-1, lin-45 raf, let-537/mek-2 or sur-1/mpk-1 cause defects in sex myoblast final positions that resemble those seen in gonad-ablated animals, while constitutively active let-60 ras(G13E) trans-genes allow fairly precise positioning to occur in the absence of the gonad. A mosaic analysis demonstrated that let-60 ras is required within the sex myoblasts to control proper positioning. Our results suggest that gonadal signals normally stimulate let-60 ras activity in the sex myoblasts, thereby making them competent to sense or respond to positional cues that determine the precise endpoint of migration. let-60 ras may have additional roles in sex myoblast guidance as well. Finally, we have also investigated genetic interactions between let-60 ras and other genes important for sex myoblast migration, including egl-15, which encodes a fibroblast growth factor receptor tyrosine kinase (D. L. DeVore, H. R. Horvitz and M. J. Stern (1995) Cell 83, 611–623). Since mutations reducing Ras pathway activity cause a different phenotype than those reducing egl-15 activity and since constitutive Ras activity only partially suppresses the migration defects of egl-15 mutants, we argue that let-60 ras and egl-15 do not act together in a single linear pathway.

In Vitro Expression Studies of Two Mutations on the Metalloprotease and First Cub Domains of the ADAMTS-13 Gene Leading to Severe ADAMTS-13 Deficiency and Chronic Recurrent TTP.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 514-514 ◽  
Author(s):  
Flora Peyvandi ◽  
Silvia Lavoretano ◽  
Raimondo De Cristofaro ◽  
Carla Valsecchi ◽  
Giuliana Merati ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Structure Prediction ◽  
Mutational Analysis ◽  
Secondary Structure Prediction ◽  
Missense Mutations ◽  
Western Blots ◽  
Hek 293 ◽  
Cell Lysates ◽  
Expression Studies

Abstract Thrombotic thrombocytopenic purpura (TTP) is associated with a severe deficiency of ADAMTS-13, a metalloprotease that processes physiologically the hyperactive ultralarge multimers of von Willebrand factor (VWF). We report the molecular characterization of a congenital case of TTP with < 6% ADAMTS-13 activity and no ADAMTS-13 inhibitor in plasma. Mutational analysis showed heterozygosity for 2 missense mutations: a G to A substitution at nucleotide 262 in exon 3 (Val88Met), in the metalloprotease domain of ADAMTS-13 and a substitution of G to T at nucleotide 3707 in exon 27 (Gly1239Val), in the first CUB domain. To explore the mechanism of the defect, the wild type protease (ADAMTS-13-WT) and each mutant (ADAMTS-13-Val88Met, ADAMTS-13-Gly1239Val) were transiently expressed in HEK 293 and COS-7 cells. WT and mutant recombinant proteins (rADAMTS-13) was analysed in conditioned medium and cell lysates by western blot analysis using anti-ADAMTS-13 monoclonal and polyclonal antibodies. The enzymatic activity of the expressed rADAMTS-13 forms was evaluated by measuring the extent of VWF multimer degradation with a quantitative immunoblot assay. Immunohistochemistry experiments were performed to study the transport of the recombinant protein between the ER and the Golgi complex. A secondary structure prediction (SSP) was also carried out to predict the effect of these mutations on the ADAMTS-13 structure. Western blots showed a dense band in the medium with a molecular weight of ~190 Kda, corresponding to the secreted rADAMTS-13-WT protein. The medium of the cells transfected with ADAMTS-13-Val88Met showed a fainter band roughly estimated to be 40–50% of the WT band. rADAMTS-13-Gly1239Val was not detectable in the medium. Both mutants were detected in cell lysates in amounts similar to ADAMTS-13-WT. rADAMTS-13 WT was able to degrade of HMW multimers, whereas rADAMTS-13 Val88Met as the well as media of cells cotrasfected by both mutant constructs lacked enzymatic activity, recapitulating the patient’s plasma pattern. By immunohistochemistry rADAMTS-13-WT was mainly localized in the perinuclear area, whereas rADAMTS-13-Gly1239Val was diffused throughout the cytoplasm with no perinuclear enhancement. rADAMTS-13-Val88Met showed increased staining in the cytoplasm but also a perinuclear enhancement. SSP analysis showed no change in the helical region including Gly1239 following replacement with Val. We hypothesize that Gly1239Val may directly alter molecular recognition, leading to a secretion pathway alteration and to retention of the mutant protein in the ER compartment. SSP analysis also showed that the Val88Met mutation, located in the metalloprotease domain, may enhance the conformational instability of the 80–87 region containing Glu83 involved in a Ca++ ion coordination. This effect may be responsible for both the observed partial secretion alteration and enzymatic activity defect of the mutant. These expression studies explain how these two gene mutations together lead to the absence of ADAMTS-13 enzymatic activity in patient’s plasma.

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