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M I Simon

Publications and source records attributed to M I Simon.

At least 55 records · Page 3Linked to original sources
PubMed1998

Autophosphorylation and ADP regulate the Ca2+-dependent interaction of recoverin with rhodopsin kinase.

Recoverin is a 23 kDa myristoylated Ca2+-binding protein that inhibits rhodopsin kinase. We have used surface plasmon resonance to investigate the influences of Ca2+, myristoylation, and adenine nucleotides on the recoverin-rhodopsin kinase interaction. Our analyses confirmed that Ca2+ is required for recoverin to bind RK. Myristoylation had little effect on the affinity of recoverin for the kinase, but it raised the K0.5 for Ca2+ from 150 nM for nonacylated recoverin to 400 nM for myristoylated recoverin. Finally, our studies also revealed two separate and previously unreported effects of adenine nucleotides on the recoverin-rhodopsin kinase binding. The interaction is weakened by autophosphorylation of the kinase, and it is strengthened by the presence of ADP.

Adenosine Diphosphate↗
PubMed1998

COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans.

Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene, COS1, from the opportunistic pathogen Candida albicans by using a PCR-based screening strategy. The sequence of COS1 indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus Neurospora crassa. COS1 is also identical to a gene called CaNIK1 identified in C. albicans by low stringency hybridization using CaSLN1 as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998) Microbiol. 44, 425-432]. We assess the function of COS1/CaNIK1 by constructing a diploid deletion mutant. Mutants lacking both copies of COS1 appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the Deltanik-1 mutant, the Deltacos1/Deltacos1 mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both Deltanik-1 and Deltacos1/Deltacos1 mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.

Amino Acid Sequence↗
PubMed1998

Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability.

The crystal structure of CheY protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.

Amino Acid Sequence↗
PubMed1998

Patterns of expression for the mRNA corresponding to the four isoforms of phospholipase Cbeta in mouse brain.

Ligand binding to neurotransmitter and hormone receptors which couple to the Gq subclass of GTP-binding protein leads to the activation of phospholipase Cbeta (PLCbeta) which hydrolyses phosphatidyl-inositol 4,5-bisphosphate, yielding a pair of second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3). The expression of PLCbeta1-4 mRNAs was comparatively examined by in situ hybridization in the mouse brain. In adults, PLCbeta1 mRNA was expressed predominantly in the telencephalon, including the cerebral cortex, hippocampus, amygdala, lateral septum and olfactory bulb, with little expression in most thalamic nuclei. PLCbeta2 mRNA was distributed in the white matter, suggesting its expression in non-neuronal cells, most likely oligodendrocytes. PLCbeta3 mRNA was specifically expressed in cerebellar Purkinje cells. The highest levels of PLCbeta4 mRNA were detected in Purkinje cells. High levels of PLCbeta4 mRNA were also found in the thalamus and medial septum, whereas weak signals were detected in most telencephalic regions, thus showing an expression pattern almost reciprocal to that of PLCbeta1 mRNA. During development, such characteristic regional expression of PLCbeta1 and PLCbeta4 were observed starting in late foetal stages, while specific expression of PLCbeta2 and PLCbeta3 appeared in early postnatal stages. We conclude that despite the existence of four PLCbeta isoforms, only one or two of them is expressed in individual neurons and glial cells. The distinct expression of PLCbetas provides a molecular basis for analysing the nature of the specific signal transduction pathway leading to the production of diacylglycerol and IP3 in distinct cell types and in different regions of the brain.

Aging↗
PubMed1997

Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking Galphaq.

Mice lacking the alpha-subunit of the heterotrimeric guanine nucleotide binding protein Gq (Galphaq) are viable but suffer from ataxia with typical signs of motor discoordination. The anatomy of the cerebellum is not overtly disturbed, and excitatory synaptic transmission from parallel fibers to cerebellar Purkinje cells (PCs) and from climbing fibers (CFs) to PCs is functional. However, about 40% of adult Galphaq mutant PCs remain multiply innervated by CFs because of a defect in regression of supernumerary CFs in the third postnatal week. Evidence is provided suggesting that Galphaq is part of a signaling pathway that is involved in the elimination of multiple CF innervation during this period.

Age Factors↗
PubMed1997

Prolonged photoresponses in transgenic mouse rods lacking arrestin.

Arrestins are soluble cytoplasmic proteins that bind to G-protein-coupled receptors, thus switching off activation of the G protein and terminating the signalling pathway that triggers the cellular response. Although visual arrestin has been shown to quench the catalytic activity of photoexcited, phosphorylated rhodopsin in a reconstituted system, its role in the intact rod cell remains unclear because phosphorylation alone reduces the catalytic activity of rhodopsin. Here we have recorded photocurrents of rods from transgenic mice in which one or both copies of the arrestin gene were disrupted. Photoresponses were unaffected when arrestin expression was halved, indicating that arrestin binding is not rate limiting for recovery of the rod photoresponse, as it is in Drosophila. With arrestin absent, the flash response displayed a rapid partial recovery followed by a prolonged final phase. This behaviour indicates that an arrestin-independent mechanism initiates the quench of rhodopsin's catalytic activity and that arrestin completes the quench. The intensity dependence of the photoresponse in rods lacking arrestin further suggests that, although arrestin is required for normal signal termination, it does not participate directly in light adaptation.

Adaptation, Ocular↗
PubMed1997

Constitutively active Galphaq and Galpha13 trigger apoptosis through different pathways.

We investigated the effect of expression of constitutively active Galpha mutants on cell survival. Transfection of constitutively active Galphaq and Galpha13 in two different cell lines caused condensation of genomic DNA and nuclear fragmentation. Endonuclease cleavage of genomic DNA was followed by labeling the DNA fragments and subsequent flow cytometric analysis. The observed cellular phenotype was identical to the phenotype displayed by cells undergoing apoptosis. To distinguish between the apoptosis-inducing ability of the two Galpha-subunits, the signaling pathways involved in this cellular function were investigated. Whereas Galphaq induced apoptosis via a protein kinaseC-dependent pathway, Galpha13 caused programmed cell death through a pathway involving the activation of the small G-protein Rho. Both of the pathways leading to apoptosis were blocked by overexpression of bcl-2. In contrast to other apoptosis-inducing systems, expression of constitutively active Galphaq and Galpha13 triggered apoptosis in high serum as well as in defined medium.

Animals↗
PubMed1997

Defective platelet activation in G alpha(q)-deficient mice.

Platelets are small disc-shaped cell fragments which undergo a rapid transformation when they encounter vascular damage. They become more spherical and extrude pseudopodia, their fibrinogen receptors are activated, causing them to aggregate, they release their granule contents, and eventually form a plug which is responsible for primary haemostasis. Activation of platelets is also implicated in the pathogenesis of unstable angina, myocardial infarction and stroke. Here we show that platelets from mice deficient in the alpha-subunit of the heterotrimeric guanine-nucleotide-binding protein Gq are unresponsive to a variety of physiological platelet activators. As a result, G alpha(q)-deficient mice have increased bleeding times and are protected from collagen and adrenaline-induced thromboembolism. We conclude that G alpha(q) is essential for the signalling processes used by different platelet activators and that it cannot be replaced by G alpha(i) or the beta gamma subunits of the heterotrimeric G proteins. G alpha(q) may thus be a new target for drugs designed to block the activation of platelets.

Animals↗
PubMed1997

Roles of phospholipase C beta2 in chemoattractant-elicited responses.

The physiological roles of phospholipase C (PLC) beta2 in hematopoiesis, leukocyte function, and host defense against infection were investigated using a mouse line that lacks PLC beta2. PLC beta2 deficiency did not affect hematopoiesis, but it blocked chemoattractant-induced Ca2+ release, superoxide production, and MAC-1 up-regulation in neutrophils. In view of these effects, it was surprising that the absence of PLC beta2 enhanced chemotaxis of different leukocyte populations and sensitized the in vivo response of the PLC beta2-deficient mice to bacteria, viruses, and immune complexes. These data raise questions about the roles that PLC beta2 may play in signal transduction induced by chemoattractants in leukocytes.

Animals↗
PubMed1997

Characterization of a Goalpha mutant that binds xanthine nucleotides.

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goalpha mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goalpha subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoalphaD273N/Q205L (GoalphaX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoalphaX (GoalphaD273N/Q205L) formed heterotrimers with betagamma complexes only in the presence of xanthine diphosphate (XDP), and the binding to betagamma was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoalphaX protein underwent a conformational change similar to that of the activated wild-type Goalpha. In transfected COS-7 cells, we demonstrate that the interaction between GoalphaX and betagamma occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein alpha subunit.

Amino Acid Sequence↗
PubMed1997

Uncoupled phosphorylation and activation in bacterial chemotaxis. The 2.3 A structure of an aspartate to lysine mutant at position 13 of CheY.

An aspartate to lysine mutation at position 13 of the chemotaxis regulatory protein CheY causes a constitutive tumbly phenotype when expressed at high copy number in vivo even though the mutant protein is not phosphorylatable. These properties suggest that the D13K mutant adopts the active, signaling conformation of CheY independent of phosphorylation, so knowledge of its structure could explain the activation mechanism of CheY. The x-ray crystallographic structure of the CheY D13K mutant has been solved and refined at 2.3 A resolution to an R-factor of 14.3%. The mutant molecule shows no significant differences in backbone conformation when compared with the wild-type, Mg2+-free structure, but there are localized changes within the active site. The side chain of lysine 13 blocks access to the active site, whereas its epsilon-amino group has no bonding interactions with other groups in the region. Also in the active site, the bond between lysine 109 and aspartate 57 is weakened, and the solvent structure is perturbed. Although the D13K mutant has the inactive conformation in the crystalline form, rearrangements in the active site appear to weaken the overall structure of that region, potentially creating a metastable state of the molecule. If a conformational change is required for signaling by CheY D13K, then it most likely proceeds dynamically, in solution.

Amino Acid Sequence↗
PubMed1997

The retinal specific protein RGS-r competes with the gamma subunit of cGMP phosphodiesterase for the alpha subunit of transducin and facilitates signal termination.

In vertebrate photoreceptor cells, transducin mediates signaling between rhodopsin and cGMP phosphodiesterase by transiently binding its gamma subunit (PDEgamma). For the termination of signaling GTP hydrolysis by the transducin alpha subunit (TDalpha) GTPase is required. This reaction can be accelerated by GTPase-activating proteins (GAPs), e.g. PDEgamma. Recently we identified a second retinal GAP that interacts with TDalpha, RGS-r. Here we compare the GAP function of RGS-r and PDEgamma. Both proteins stimulated single turnover GTPase of TDalpha; however, RGS-r was more effective than PDEgamma. When added together, PDEgamma competitively inhibited the RGS-r-stimulated GTPase. In addition, the interaction of TDalpha in its GTP-bound form (TDalphaGTPgammaS), the transition state (TDalphaGDP*AMF) and the GDP-bound form (TDalphaGDP) with RGS-r and PDE, respectively, was measured by surface plasmon resonance. PDEgamma displayed highest affinity for TDalphaGTPgammaS, weaker affinity for TDalphaGDP*AMF, and weakest affinity for TDalphaGDP. RGS-r exhibited only a comparable high affinity for TDalphaGDP*AMF. We conclude that the observed competition between RGS-r and PDEgamma for TDalpha occurs when the hydrolysis of GTP is initiated. By competing with PDEgamma and removing it from TDalpha as well as increasing Pi release, RGS-r apparently facilitates signal termination and TDalpha recycling.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Vascular system defects and impaired cell chemokinesis as a result of Galpha13 deficiency.

Heterotrimeric GTP-binding proteins (G proteins) participate in cellular signaling and regulate a variety of physiological processes. Disruption of the gene encoding the G protein subunit alpha13 (Galpha13) in mice impaired the ability of endothelial cells to develop into an organized vascular system, resulting in intrauterine death. In addition, Galpha13 (-/-) embryonic fibroblasts showed greatly impaired migratory responses to thrombin. These results demonstrate that Galpha13 participates in the regulation of cell movement in response to specific ligands, as well as in developmental angiogenesis.

Animals↗
PubMed1997

A fosmid-based genomic map and identification of 474 genes of the hyperthermophilic archaeon Pyrobaculum aerophilum.

We have constructed a physical map of the approximately 1.7-Mb genome of the hyperthermophilic archaeon Pyrobaculum aerophilum. Derived from a 12x coverage genomic fosmid library with an average insert size of 36 Kb, the map consists of a single circular contig of 96 overlapping fosmid clones with 211 markers ordered along them. One hundred of the sequence markers have strong similarities to known genes. Many overlaps were also checked using restriction fingerprint analysis. This map is an important step in the elucidation of the sequence of the entire genome of Pyrobaculum aerophilum. To this end we have determined more than 95% of the genome with 15,000 random sequences. Each sequence has been screened against the public sequence databases to identify similarities to known genes. We report here a list of the 474 putative genes we have identified.

Animals↗
PubMed1997

Analysis of the 1.1-Mb human alpha/delta T-cell receptor locus with bacterial artificial chromosome clones.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents. The 1.1-Mb alpha/delta T-cell receptor locus of humans was mapped and partially sequenced with BAC clones. Seventeen BAC clones covered the 1.1-Mb alpha/delta locus, with the exception of one small gap that was expected from the coverage that a 3.7-fold BAC library is likely to provide. The end sequences of the BAC inserts could be obtained directly from the BAC DNA by sequencing with the chain terminator chemistry. Five complete BAC inserts were sequenced directly by the shotgun approach. The ends of the 17 BAC inserts were distributed evenly across the locus. By several independent criteria, the BAC clones faithfully represented the genomic DNA, with the exception of a single clone with a 68-kb deletion. These BAC features led to the proposal of a new approach to sequence the human genome.

Bacteriophage M13↗