Biomedical subjects

M N Simon

Publications and source records attributed to M N Simon.

At least 37 records · Page 2Linked to original sources

PubMed1995

Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway.

In the budding yeast Saccharomyces cerevisiae, the process of conjugation of haploid cells of genotype MATa and MAT alpha to form MATa/alpha diploids is triggered by pheromones produced by each mating type. These pheromones stimulate a cellular response by interaction with receptors linked to a heterotrimeric G protein. Although genetic analysis indicates that the pheromone signal is transmitted through the G beta gamma dimer, the initial target(s) of G protein activation remain to be determined. Temperature-sensitive cells with mutations of the CDC24 and CDC42 genes, which are incapable of budding and of generating cell polarity at the restrictive temperature, are also unable to mate. Cdc24 acts as a guanylyl-nucleotide-exchange factor for the Rho-type GTPase Cdc42, which has been shown to be a fundamental component of the molecular machinery controlling morphogenesis in eukaryotic cells. Therefore, the inability of cdc24 and cdc42 mutants to mate has been presumed to be due to a requirement for generation of cell polarity and related morphogenetic events during conjugation. But here we show that Cdc42 has a direct signalling role in the mating-pheromone response between the G protein and the downstream protein kinase cascade.

Cell Cycle Proteins↗

PubMed1995

A protein kinase from Dictyostelium discoideum with an unusual acidic repeat domain.

DdKinX codes for 1093 amino acids which are organized in four regions: the N-terminal catalytic domain, a region containing 30% acidic amino acids, tandem repeats of the motif VKVEEPVEE and the C-terminus. Identity with other protein kinases is 25 to 30%. Descendent trees show that DdKinX does not belong to any of the known kinase branches.

Amino Acid Sequence↗

PubMed1995

Site-directed photo-cross-linking of rRNA transcription initiation complexes.

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

Acanthamoeba↗

PubMed1994

Filamentous hemagglutinin of Bordetella pertussis. A bacterial adhesin formed as a 50-nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns.

The filamentous hemagglutinin (FHA) of Bordetella pertussis is an adhesin that binds the bacteria to cells of the respiratory epithelium in whooping-cough infections. Mature FHA is a 220 kDa secretory protein that is highly immunogenic and has been included in acellular vaccines. We have investigated its structure by combining electron microscopy and circular dichroism spectroscopy (CD) with computational analysis of its amino acid sequence. The FHA molecule is 50 nm in length and has the shape of a horseshoe nail: it has a globular head that appears to consist of two domains; a 35 nm-long shaft that averages 4 nm in width, but tapers slightly from the head end; and a small, flexible, tail. Mass measurements by scanning transmission electron microscopy establish that FHA is a monomer. Its sequence contains two regions of tandem 19-residue pseudo-repeats: the first, of 38 cycles, starts at residue 344; the second, of 13 cycles, starts at residue 1440. The repeat motifs are predicted to consist of short beta-strands separated by beta-turns, and secondary structure measurements by CD support this prediction. We propose a hairpin model for FHA in which the head is composed of the terminal domains; the shaft consists mainly of the repeat regions conformed as amphipathic, hyper-elongated beta-sheets, with their hydrophobic faces apposed; and the tail is composed of the intervening sequence. Further support for the model was obtained by immuno-labeling electron microscopy. The 19-residue repeats of FHA have features in common with the leucine-rich repeats (LRRs) that are present in many eukaryotic proteins, including some adhesion factors. The model is also compared with the two other classes of filamentous proteins that are rich in beta-structure, i.e. viral adhesins and two beta-helical secretory proteins. Our proposed structure implies how the functionally important adhesion sites and epitopes of FHA are distributed: its tripeptide (RGD) integrin-binding site is assigned to the tail; the putative hemagglutination site forms part of the head; and two classes of immunodominant epitopes are assigned to opposite ends of the molecule. Possible mechanisms are discussed for two modes of FHA-mediated adhesion.

Adhesins, Bacterial↗

PubMed1993

Regulation of Dictyostelium morphogenesis by cAMP-dependent protein kinase.

During formation of the Dictyostelium slug extracellular cAMP signals direct the differentiation of prespore cells and DIF, a chlorinated hexaphenone, induces the differentiation of prestalk cells. At culmination the slug transforms into a fruiting body, composed of a stalk supporting a ball of spores. A dominant inhibitor of cAMP-dependent protein kinase (PKA) expressed under the control of a prestalk-specific promoter blocks the differentiation of prestalk cells into stalk cells. Analysis of a gene specifically expressed in stalk cells suggests that PKA acts to remove a repressor that prevents the premature induction of stalk cell differentiation by DIF during slug migration. PKA is also necessary for the morphogenetic movement of prestalk cells at culmination. Expression of the PKA inhibitor under control of a prespore-specific promoter blocks the accumulation of prespore mRNA sequences and prevents terminal spore cell differentiation. Thus PKA is essential for progression along both pathways of terminal differentiation but with different mechanisms of action. On the stalk cell pathway it acts to regulate the action of DIF while on the spore cell pathway PKA itself seems to act as the inducer of spore cell maturation. Ammonia, the extracellular signal which regulates the entry into culmination, acts by controlling the intracellular concentration of cAMP and thus exerts its effects via PKA. The fact that PKA is necessary for both prespore and spore gene expression leads us to postulate the existence of a signalling mechanism which converts the progressive rise in cAMP concentration during development into discrete, PKA-regulated gene activation events.

Animals↗

PubMed1993

The short tail-fiber of bacteriophage T4: molecular structure and a mechanism for its conformational transition.

Electron microscopy, image processing and computational sequence analysis were used to investigate the structure of the short tail-fiber of bacteriophage T4. This molecule, an oligomer of gp12, is an adhesin that binds the virion irreversibly to the bacterial surface. Short tail-fibers were isolated from mutant-infected cells in which gp12 is synthesized and assembled correctly, but not incorporated into virions. Visualized in negative stain, these filamentous molecules are approximately 38 nm in total length, with an arrowhead-shaped head (approximately 10 nm long by 6 nm wide), a 24-nm shaft of uniform width (approximately 3.8 nm), and a small, seemingly flexible, tail. The primary sequence contains a domain consisting of tandem quasi-repeats, each about 40 residues long, extending from approximately residue 50 to residue 320. Molecular mass analyses by scanning transmission electron microscopy confirm that the molecule is a trimer. The masses of the head, shaft, and tail domains are consistent with (trimers of) the carboxy-terminus, the repeat region, and the amino-terminus, respectively. When short tail-fibers are visualized extending from baseplates, their heads are distal, i.e., detached, implying that it is the tail that remains in contact with the baseplate. Analysis of the molecules' curvature properties detects three hinge-sites: these suggest how the short tail-fiber may be initially accommodated in a compact conformation in the "hexagon" state of the baseplate, from which it converts to the extended conformation when the baseplate switches into its "star" state.

Amino Acid Sequence↗

PubMed1993

Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii.

Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases.

Blastocladiella↗

PubMed1993

Interacting signalling pathways regulating prestalk cell differentiation and movement during the morphogenesis of Dictyostelium.

Analysis of the expression patterns of two genes encoding extracellular matrix proteins shows there to be an unexpectedly complex pattern of prestalk cell differentiation and movement during the morphogenesis of Dictyostelium. The organism employs both cell sorting and positional differentiation to generate a patterned structure but these two mechanisms are used at different times during development. During slug formation prestalk cells arise at scattered positions within the aggregate and then move to its apex to form the tip. In contrast, during culmination, stalk cell differentiation occurs in a positionally localized manner at the entrance to the stalk tube. Two interacting signalling pathways regulate the differentiation of prestalk and stalk cells. Prestalk cell differentiation is induced by DIF, a chlorinated hexaphenone, and a repression mechanism prevents DIF acting to induce premature stalk cell differentiation during slug migration. At culmination intracellular cAMP levels rise, the cAMP dependent protein kinase (PKA) is activated and the block to stalk cell differentiation is lifted. Activation of PKA is also necessary in order that prestalk cells move to the entrance of the stalk tube at culmination. Thus, in Dictyostelium, PKA plays a role both in the regulation of cellular differentiation and in morphogenetic cell movement.

Animals↗

PubMed1992

Mass determination and estimation of subunit stoichiometry of the bacterial hook-basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy.

The basal body, a part of the rotary motor of the bacterial flagellum, is a multiprotein assembly that consists of four rings (denoted M, S, P, and L) and an axial rod (denoted R). From analysis of scanning transmission electron microscopy images of hook-basal body preparations isolated from Salmonella typhimurium, we have determined the masses of the basal body and three of its subcomplexes. The mass of the basal body (i.e., the four rings and rod) is 4400 +/- 490 kDa (mean +/- SD; n = 54). The mass of the LPR subcomplex (i.e., L and P rings and the whole rod) is 2600 +/- 380 kDa (n = 55), that of the L and P rings and the distal part of the rod is 2100 +/- 320 kDa (n = 25), and the mass of the L and P ring subcomplex is 1700 +/- 260 kDa (n = 514). These results, together with the masses of the component proteins, indicate that the rings contain approximately 26 subunits each and that the mass of the rod is consistent with a composition of approximately 6 copies each of three of the rod proteins FlgB, FlgC, and FlgF and approximately 26 copies of FlgG as determined by Jones et al. [Jones, C. J., Macnab, R. M., Okino, H. & Aizawa, S.-I. (1990) J. Mol. Biol. 212, 377-387] using quantitative gel electrophoresis. The results of Jones et al., together with ours, account for all proteins in the basal body to within approximately 5% (or 200 kDa).

Bacterial Proteins↗

PubMed1992

Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase.

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.

Animals↗

PubMed1992

Serine/threonine protein phosphatases in Dictyostelium discoideum: no evidence for type I activity.

Extracts from Dictyostelium discoideum contain type 2A and 2C serine/threonine-specific protein phosphatases with properties very similar to those from mammals according to their sensitivity to okadaic acid and to their dependence for divalent cations. In contrast, no type 1 protein phosphatase is found at any time of development, neither in the cytosolic nor in the particulate fraction, using glycogen phosphorylase a, casein, histone or the non-proteinous 4-Methylumbelliferyl phosphate as substrates. Both type 2A and 2C protein phosphatase activities remain constant throughout the development cycle.

Animals↗

PubMed1992

Mutation of protein kinase A causes heterochronic development of Dictyostelium.

In heterochronic mutants the relative timing of developmental events is altered compared with the wild type. This generally results in a disordered embryo, though heterochronic mutations may also be an important source of evolutionary variation. In the rapidly developing (rde) mutants of Dictyostelium, stalk and spore cells differentiate before morphogenesis is complete. We have traced the lesion in one class of these mutants to the regulatory subunit of cyclic AMP-dependent protein kinase (pk-A). Inactivation of this protein results in the unrestrained activity of the catalytic subunit, so prematurely triggering terminal cell differentiation.

Amino Acid Sequence↗

PubMed1992

Multiple roles for cAMP-dependent protein kinase during Dictyostelium development.

The cAMP-dependent protein kinase (PKA) holoenzyme of Dictyostelium comprises a single regulatory (R) and catalytic (C) subunit, and both proteins increase in concentration during cellular aggregation. In order to determine the role of the kinase, we have constructed mutants of the R subunit that are defective in cAMP binding, in inhibition of the C subunit, or in both functions. Analysis of these mutants suggests that overexpression of the unmutated R subunit, which is known to block development, occurs by direct inactivation of the C subunit rather than by an effect on intracellular cAMP levels. Cells with an inactive C subunit (PKA- cells) are defective in cAMP relay, the production of cAMP in response to extracellular cAMP stimulation. This presumably accounts for their inability to undertake aggregation. When mixed with wild-type cells, PKA- cells migrate toward the signalling centre but remain confined to the periphery of the tight aggregate and are lost from the back of the migratory slug. This suggests that PKA may be required during the late, multicellular stages of development. Consistent with this, we find that a number of postaggregative genes are not expressed in PKA- cells, even when they are allowed to synergise with normal cells.

Animals↗

PubMed1992

The mass of the Sindbis virus nucleocapsid suggests it has T = 4 icosahedral symmetry.

The mass of the nucleocapsid of the alphavirus Sindbis was determined by scanning transmission electron microscopy. The nucleocapsid was found to have an average mass of 10.54 x 10(6) Da with the largest percentage of particles showing a mass of 10.7 to 10.9 x 10(6) Da. These data predict that the nucleocapsid contains 240 copies of protein and is a T = 4 icosahedron.

Capsid↗

PubMed1991

Export of infectious particles by Escherichia coli transfected with the RF DNA of Pf1, a virus of Pseudomonas aeruginosa strain K.

Pf1 is a filamentous, single-stranded DNA virus that has Pseudomonas aeruginosa (strain K) as host. It is the longest of the filamentous bacterial viruses, and the DNA within it has the most extended conformation known. Pf1 virus cannot infect Escherichia coli (strain MM294) cells, but when these cells are transfected with the double-stranded replicative form of Pf1 DNA (RF DNA, 7.35 kb), they export low levels of infectious particles that create plaques on lawns of P. aeruginosa. Several different structural species, at least two of which are infectious, are exported. One of them, called Epf1, has virtually the same structure as Pf1, but the amount of Epf1 exported by E. coli is 10(4) lower than the amount of Pf1 exported by P. aeruginosa. The results imply that host factors affect not only the efficiency of virus assembly and export, but also the actual structures of the species exported.

Bacteriophages↗

PubMed1989

Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.

Blotting, Western↗

PubMed1988

Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones.

lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells. Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit. The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction. The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence. The results show that the Dictyostelium R subunit can be functionally expressed in E. coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein. In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit. One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D. discoideum.

Amino Acid Sequence↗

PubMed1988

cAMP-dependent protein kinase from Dictyostelium discoideum.

The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins.

Amino Acid Sequence↗