PubMed Health⌕ Search

Biomedical subjects

D A Towler

Publications and source records attributed to D A Towler.

31 records · Page 2Linked to original sources
PubMed1994

Msx-2/Hox 8.1: a transcriptional regulator of the rat osteocalcin promoter.

We recently defined an element (ACTAATTGG) within the rat osteocalcin (OC) promoter at -84 to -92 which provides approximately 70% of basal promoter activity in osteoblastic cell lines and binds a specific nuclear factor found in OC-producing ROS 17/2.8 osteosarcoma cells. Since this element closely resembles the recently described Msx-1 (Hox 7.1) homeodomain DNA binding cognate, we examined rodent osteoblastic cells lines for expression of Msx homeodomain-encoding messages. We have found and cloned a cDNA for rat Msx-2 (Hox 8.1) from a ROS 17/2.8 library and detect high levels of expression in various osteoblastic cell lines (ROS 17/2.8, RCT3, RCT1) as well as in culture passage 3 neonatal rat calvarial osteoblastic cells. Little to no expression was detected in phenotypically immature MC3T3E1 osteoblastic cells or in a variety of nonosteoblastic (ROS 25/1, C2C12, TRAB 11) mesenchymal cell lines. Dexamethasone (DEX) down-regulates Msx-2 message levels in both RCT3 and ROS 17/2.8 cells. Recombinant rat Msx-2 homeodomain expressed in Escherichia coli as a glutathione-S-transferase fusion protein binds to the rat OC promoter region -74 to -100 as determined by gel shift analysis. Recognition is dependent upon the intact ACTAATTGG motif at -84 to -92. In transient cotransfection assays using MC3T3E1 cells (which expresses very little or no endogenous Msx-2), Msx-2 suppresses the rat OC promoter 2- to 3-fold via the Msx-2 binding motif at -84 to -92. However, in ROS 17/2.8 cells, where a high level of endogenous Msx-2 mRNA is present, expression of exogenous Msx-2 does not suppress the rat OC promoter; surprisingly, Msx-2 further augments basal promoter activity by approximately 50-70%, again dependent upon the ACTAATTGG motif at -84 to -92. These data directly demonstrate that the Msx-2 homeodomain binds the rat OC promoter and that Msx-2 can act as a sequence-specific transcriptional regulator of the rat OC promoter in cultured osteoblastic cell lines. This activity is dependent upon the specific osteoblastic cellular context, similar to previous observations in nonosseous systems with other homeodomain transcription factors. These data suggest that Msx-2 may play a role in the transcriptional regulation of the osteoblast phenotype during development in the morphogenetic fields where it is expressed.

Animals↗
PubMed1994

Characterization of retinoic acid- and cell-dependent sequences which regulate zif268 gene expression in osteoblastic cells.

We have previously shown that retinoic acid (RA) induces differentiation in an osteoblastic cell line derived from embryonic rat calvaria and that RA has selective effects on zif268 gene expression in these preosteoblastic cells,distinct from those in more mature osteoblasts. In this study we demonstrate that the RA-dependent transcriptional increase in zif268 gene expression is mediated by the interaction of RA receptors (RARs) with a 17 base pair sequence in the zif268 promoter containing a single half-site motif (GTTCA), identical to each of the direct repeats seen in the RAR beta 2 gene. The sequence appears relatively RA-specific, since the zif268 RA-responsive element is not activated by 1,25-dihydroxyvitamin D3 or thyroid hormone (T3). However, cotransfection of RAR expression vectors and an SV-40 promoter chloramphenicol acetyltransferase (CAT) construct containing the single zif268 RA-responsive motif into CV-1 cells demonstrates that the alpha-, beta-, and gamma-RARs transactivate through this element. Extensive mutagenesis of the zif268 promoter region containing the RA response element (RARE) motif confirms that the transactivation and nuclear protein binding activity of this region requires only the half-site motif. The direct involvement of RAR in this DNA-protein interaction has been demonstrated by competitive gel retardation analysis using consensus RAREs and super-shifting of the DNA-protein complex with mouse alpha- or gamma-RAR monoclonal antibodies. In addition, we found that cell-specific suppression of RA-stimulated zif268 gene expression can be attributed to a 29 base pair nucleotide sequence, located downstream of the RA-responsive region in the zif268 gene. This sequence appears to be bound specifically by nuclear protein(s) from several cell types, including osteoblasts. The presence of this sequence in cis to the zif268 RARE or the consensus beta RARE completely blocks the RA-responsiveness of the zif268 gene in differentiated osteoblasts. These data extend the broad spectrum of RA-responsive sequences necessary for DNA binding and transactivation to include regulation via single RARE half-site motifs and suggest that the lack of RA responsiveness in differentiated osteoblasts may be mediated by cell-specific suppression of gene expression.

Animals↗
PubMed1994

Activity of the rat osteocalcin basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs.

A detailed analysis of the transcriptional machinery responsible for osteoblast-specific gene expression should provide tools useful for understanding osteoblast commitment and differentiation. We have defined three cis-elements important for basal activity of the rat osteocalcin (OC) promoter, located at about -200 to -180, -170 to -138, and -121 to -64 relative to the transcription initiation site. A motif (TCTGATTGTGT) present in the region between -200 and -170 that binds a multisubunit CP1/NFY/CBF-like CAAT factor complex contributes significantly to high level basal activity and presumably functions as the CAAT box for the rat OC promoter. We show that the region -121 to 32 is sufficient to confer osteoblastic cell type specificity in transient transfection assays of cultured cell lines using luciferase as a reporter. The basal promoter is active in rodent osteoblastic cell lines, but not in rodent fibroblastic or muscle cell lines. Although the rat OC box (-100 to -74) contains a CAAT motif, we could not detect CP1-like CAAT factor binding to this region. In fact, we demonstrate that a Msx-1 (Hox 7.1) homeodomain binding motif (ACTAATTG; bottom strand) in the 3'-end of the rat OC box is necessary for high level activity of the rat OC basal promoter in osteoblastic cells. A nuclear factor that recognizes this motif appears to be present in osteoblastic ROS 17/2.8 cells, which produce OC, but not in fibroblastic ROS 25/1 cells, which fail to express OC. This ROS 17/2.8 nuclear factor also recognizes the A/T-rich DNA cognates of the homeodomain-containing POU family of transcription factors. Taken together, these data suggest that a ubiquitous CP1-like CAAT factor and a cell type-restricted homeodomain containing (Msx or POU family) transcription factor interact with the proximal rat OC promoter to direct appropriate basal OC transcription in osteoblastic cells.

3T3 Cells↗
PubMed1993

Mechanism of awareness of hypoglycemia. Perception of neurogenic (predominantly cholinergic) rather than neuroglycopenic symptoms.

We sought 1) to determine which symptoms of hypoglycemia are reproducible, 2) to pharmacologically distinguish neurogenic (autonomic) from neuroglycopenic symptoms, and 3) to test the hypothesis that awareness of hypoglycemia is the result of perception of neurogenic rather than neuroglycopenic symptoms. Awareness of hypoglycemia and 19 symptoms were quantitated in 10 normal, young adults, each studied on four occasions in random sequence, during 1) clamped euglycemia (approximately 5 mM), 2) clamped hypoglycemia (approximately 2.5 mM), 3) clamped hypoglycemia with combined alpha- and beta-adrenergic blockade (phentolamine and propranolol), and 4) clamped hypoglycemia with pan-autonomic blockade (phentolamine, propranolol and atropine). Significant (ANOVA, P < 0.001) treatment effects on the awareness of hypoglycemia ("blood sugar low") were noted. No change occurred in the score for this during euglycemia, but the mean +/- SE increase was 2.1 +/- 0.4 during hypoglycemia. This increase was not reduced significantly by adrenergic blockade (1.6 +/- 0.5), but was reduced significantly and substantially (approximately 70%) by pan-autonomic blockade (0.6 +/- 0.3). Significant neurogenic symptoms included shaky/tremulous (P < 0.001), heart pounding (P < 0.001), and nervous/anxious (P = 0.002), all adrenergic; and sweaty (P < 0.001), hungry (P < 0.001), and tingling (P = 0.009), all cholinergic. Significant neuroglycopenic symptoms, those produced by hypoglycemia but not reduced by pan-autonomic blockade, included warm (P < 0.001), weak (P = 0.011), difficulty thinking/confused (P = 0.004), and tired/drowsy (P = 0.003). We conclude that muscarinic cholinergic mechanisms mediate an important and previously uncharacterized component of the neurogenic symptoms of hypoglycemia and awareness of hypoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxybutyric Acid↗
PubMed1991

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli.

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates. These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.

Acyltransferases↗
PubMed1989

Disruption of the yeast N-myristoyl transferase gene causes recessive lethality.

The structural gene for N-myristoyl transferase (NMT1) has been cloned from the budding yeast Saccharomyces cerevisiae. The gene encodes a polypeptide of 455 amino acids (Mr = 52,837) that has no identifiable significant primary sequence homology with any protein in currently available databases. Overexpression of NMT activity was achieved by means of the yeast episomal plasmid YEp24 without obvious effects on growth kinetics, cell morphology, or acylprotein metabolic labeling patterns. Insertional mutagenesis of the NMT1 locus on yeast chromosome XII caused recessive lethality, indicating that this protein acyltransferase activity is necessary for vegetative cell growth.

Acyltransferases↗
PubMed1988

11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase.

The covalent attachment of myristic acid to the NH2-terminal glycine residue of proteins is catalyzed by the enzyme myristoyl CoA:protein N-myristoyltransferase (NMT). Using synthetic octapeptide substrates we have identified and characterized an NMT activity in wheat germ lysates used for cell-free translation of exogenous mRNAs. C-12 and C-14 fatty acids are efficiently transferred to the peptides by this plant NMT, but C-10 and C-16 fatty acids are not. Glycine is required as the NH2-terminal residue: peptides with an NH2-terminal alanine were not substrates. Peptides with proline, aspartic acid, or tyrosine residues adjacent to the NH2-terminal glycine were also not myristoylated. Serine in the fifth position reduced the peptide's Km up to 4000-fold. We have chemically synthesized a sulfur analogue of myristate, 11-(ethylthio)undecanoic acid. Its CoA ester is as good a substrate as myristoyl-CoA for both wheat germ and yeast NMT. Peptides linked to 11-(ethylthio)undecanoic acid are less hydrophobic than the corresponding myristoylpeptides. 11-(Ethylthio)-undecanoic acid may, therefore, help define the role of myristic acid in targeting of acyl proteins within cells.

Acyltransferases↗
PubMed1988

Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities.

A variety of eukaryotic viral and cellular proteins possesses an NH2-terminal N-myristoylglycine residue important for their biological functions. Recent studies of the primary structural requirements for peptide substrates of the enzyme responsible for this modification in yeast demonstrated that residues 1, 2, and 5 play a critical role in enzyme: ligand interactions (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon J. I. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 2708-2812). This was determined by examining as substrates a series of synthetic peptides whose sequences were systematically altered from a "parental" peptide derived from the known N-myristoylprotein bovine heart cyclic AMP-dependent protein kinase (A kinase) catalytic subunit. We have now extended these studies in order to examine structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT). The interaction between yeast NMT and the side chain of residue 5 in peptide ligands is apparently sterically constrained, since Thr5 is unable to promote the very high affinity binding observed with a Ser5 substitution. A substrate hexapeptide core has been defined which contains much of the information necessary for recognition by this lower eukaryotic NMT. Addition of COOH-terminal basic residues to this hexapeptide enhances peptide binding, while COOH-terminal acidic residues destabilize NMT: ligand interactions. Based on the results obtained from our in vitro studies of over 80 synthetic peptides and yeast NMT, we have identified a number of potential N-myristoylproteins from searches of available protein databases. These include hepatitis B virus pre-S1, human SYN-kinase, rodent Gi alpha, and bovine transducin-alpha. Peptides corresponding to the NH2-terminal sequences of these proteins and several known N-myristoylproteins were assayed using yeast NMT as well as partially purified rat liver NMT. While a number of the synthetic peptides exhibited similar catalytic properties with the yeast and mammalian enzymes, surprisingly, the SYN-kinase, Gi alpha, and transducin-alpha peptides were N-myristoylated by rat NMT but not by yeast NMT. This suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.

Acyl Coenzyme A↗
PubMed1988

Lipoproteins of Haemophilus influenzae type b.

Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.

Bacterial Outer Membrane Proteins↗
PubMed1987

Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase.

Using synthetic octapeptides, we examined the amino-terminal sequence requirements for substrate recognition by myristoyl-CoA:protein N-myristoyl transferase (NMT). NMT is absolutely specific for peptides with amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val, or Leu penultimate to the amino-terminal Gly were substrates, whereas peptides with Asp, D-Asn, Phe, or Tyr at this position were not myristoylated. Peptides with aromatic residues at this position competitively inhibited myristoylation of substrates, introducing the possibility of developing specific in vivo inhibitors of NMT. Peptides having sequences which correspond to those of known N-myristoyl proteins, including p60src, appear to be recognized by a single enzyme, and yeast and murine NMT have identical substrate specificities. The catalytic selectivity of NMT for myristoyl transfer accounts for the remarkable acyl chain specificity of this enzyme.

Acyl Coenzyme A↗
PubMed1987

Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase.

Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of approximately equal to 500 in the apparent Km in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.

Acyltransferases↗
PubMed1985

Specificity of fatty acid acylation of cellular proteins.

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.

Acylation↗