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At least 1,513 records · Page 84Linked to original sources
PubMed1997

Integrin-mediated transfection with peptides containing arginine-glycine-aspartic acid domains.

Two synthetic peptides comprising an RGD moiety for integrin binding and a polylysine moiety for DNA binding were tested for transfection efficiency under a variety of different conditions. Binding of target cells to the peptide was shown to be strongly dependent on cyclisation of the peptides via cysteine residues. Low (10 microM) concentrations of chloroquine, added to assist endocytic exit, unexpectedly reduced transfection efficiency in two of the cell lines tested. COS-7 and ECV304. However, transfection efficiency increased at higher chloroquine concentrations and exceeded that in the absence of chloroquine in the case of the COS-7 and A375M cell lines. With the ECV304 cell line, optimum transfection occurred in the absence of chloroquine. Transfection efficiency of the peptides was greatest at peptide:DNA ratios of 4:1 (w/w), which were calculated to generate complexes containing approximately 5000 peptide molecules per plasmid. This represented approximately a 6:1 ratio of positive to negative charges. Peptide 5 was shown to have a higher transfection efficiency under most conditions, possibly because of more efficient stabilisation of cyclisation by two cysteine-cysteine bonds.

Animals↗
PubMed1985

A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin.

Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin. Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp. Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide. These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin. In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin. Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin. These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities.

Amino Acid Sequence↗
PubMed1999

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins alphavbeta3 and alphaIIbbeta3.

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin alphavbeta3 (also known as the vitronectin receptor) or alphaIIbbeta3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin alphaIIbbeta3. Defined beta3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing alphavbeta3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.

Alleles↗
PubMed1996

Reengineering the nucleotide cofactor specificity of the RecA protein by mutation of aspartic acid 100.

We have recently obtained evidence for a direct linkage between the S0.5 (S0.5 is the substrate concentration required for half-maximal velocity) value of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex, with an S0.5 value of 125 microM or less required for stabilization of the strand exchange-active conformation. For example, although ATP and ITP are hydrolyzed by the RecA protein with the same turnover number (18 min-1), ATP (S0.5 = 45 microM) functions as a cofactor for the strand exchange reaction, whereas ITP (S0.5 = 500 microM) is inactive as a strand exchange cofactor. The RecA protein crystal structure suggests that cofactor specificity is determined by Asp100, which likely forms a hydrogen bond with the exocyclic 6-amino group of ATP; the higher S0. 5 value for ITP is presumably due to unfavorable interactions between Asp100 and the 6-carbonyl group of the inosine ring. To test this hypothesis, we prepared a mutant RecA protein in which Asp100 was replaced by an asparagine residue. The S0.5(ITP) for the [D100N]RecA protein is 125 microM, indicating favorable interactions between the Asn100 side chain and the 6-carbonyl group of ITP. Correspondingly, ITP functions as a cofactor for the strand exchange activity of the [D100N]RecA protein. This result demonstrates the importance of the residue at position 100 in determining nucleotide cofactor specificity and underscores the importance of the S0.5 value in the RecA protein-promoted strand exchange reaction.

Adenosine Triphosphate↗
PubMed1999

Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity.

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.

Amino Acid Sequence↗
PubMed2001

The peroxisome proliferator-induced cytosolic type I acyl-CoA thioesterase (CTE-I) is a serine-histidine-aspartic acid alpha /beta hydrolase.

Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism. We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor alpha (Hunt, M. C., Nousiainen, S. E. B., Huttunen, M. K., Orii, K. E., Svensson, L. T., and Alexson, S. E. H. (1999) J. Biol. Chem. 274, 34317-34326). Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases. In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds. The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity. Extensive sequence analysis pinpointed Ser(232), Asp(324), and His(358) as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity. A S232C mutant retained about 2% of the wild type activity and incubation with (14)C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein. These data are discussed in relation to the structure/function of acyl-CoA thioesterases versus acyltransferases. Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C(12)-C(20) acyl-CoAs.

Amino Acid Sequence↗
PubMed1986

Sequence and expression of four mutant aspartic acid tRNA genes from the mitochondria of Saccharomyces cerevisiae.

Expression of the mitochondrial tRNAAsp gene of Saccharomyces cerevisiae has been examined in five syn- mutants known to affect tRNAAsp function, and in a rho- mutant which accumulates precursor tRNAs. By comparison of wild-type versus mutant DNA sequence, the lesion in each syn- mutant has been identified as a single base change within the mitochondrial tRNAAsp structural gene. The mutant tRNAAsp genes are transcribed, and the transcripts can be processed to mature 4S-size tRNAAsp. The steady-state level of each mutant tRNAAsp is lower than that of wild-type tRNAAsp. The RNA from two of the syn- mutants contained a second, slow-migrating form of mitochondrial tRNAAsp which is correctly processed at the 5' end. We conclude that the lesions in the syn- mitochondrial tRNAAsp genes block neither transcription of these genes, nor 5'-end processing of the transcripts. The effect of each point mutation must be manifested at the level of 3'-end processing, or at a functional level.

Base Sequence↗
PubMed1980

Isolation and characterization of sulfated glycoproteins from the brush border fraction and the soluble fraction of rabbit small intestine.

The brush border fraction (Fr. P2) and the calcium chloride (10 mM)-soluble fraction (Fr. S) were separated from the small intestinal mucosa of rabbit. Sulfated glycoproteins, P-SGP and S-SGP, were then purified from Fr. P2 and Fr. S by zone electrophoresis and gel filtration, respectively. P-SGP and S-SGP were both electrophoretically homogeneous, although their mobilities differed from each other. P-SGP and S-SGP contained 61.8 and 35.% protein, 31.8 and 55.0% carbohdyrate, and 1.1 and 2.7% sulfate, respectively. The major constituent sugars in P-SGP and S-SGP were galactose and glucosamine, while mannose, sialic acid, and L-fucose were minor components. In addition, galactosamine was a major sugar in S-SGP, but a minor one in P-SGP. The major amino acids of the protein moiety of P-SGP were glutamic acid, aspartic acid, glycine, and alanine, while those of S-SGP were threonine, proline, glutamic acid, aspartic acid, serine, and glycine.

Amino Acids↗
PubMed1990

Alteration in folding efficiency and conformation of recombinant human tumor necrosis factor-alpha by replacing cysteines 69 and 101 with aspartic acid 69 and arginine 101.

An analog of human tumor necrosis factor-alpha (TNF-alpha) was created involving the replacement of Cys69 with Asp and Cys101 with Arg. The solution structure and behavior of this analog were compared with the native protein. The analog exhibited a greatly decreased folding efficiency following dilution from urea, but essentially identical circular dichroic spectra in both the folded and unfolded states. The Stokes radius of the native and analog TNF-alpha in the folded state were identical, with the analog exhibiting a slight broadening of the eluting peak. The fluorescence emission spectrum of the native protein exhibits a plateau from 320 to 328 nm, while the spectrum of the analog consisted of a single peak with a maximum at 335 nm. The analog also had a 1.4-fold increase in the fluorescence intensity. Limited proteolysis of the analog resulted in only one of the two peptides seen following digestion of the native protein, and this product was less stable than the equivalent native protein fragment. The analog exhibited a 10-fold lower cytolytic activity than the native protein. These results demonstrated that the disulfide bond is not necessary for folding and activity, but are consistent with the analog having a looser, more flexible structure in solution than the native TNF-alpha.

Amino Acid Sequence↗
PubMed1991

Soluble laminin and arginine-glycine-aspartic acid containing peptides differentially regulate type IV collagenase messenger RNA, activation, and localization in testicular cell culture.

Rat testicular cells in culture produce several metalloproteinases including type IV collagenases (Sang et al. Biol Reprod 1990; 43:946-955, 956-964). We have now investigated the regulation of testicular cell type IV collagenase and other metalloproteinases in vitro. Soluble laminin stimulated Sertoli cell type IV collagenase mRNA levels. However, three peptides corresponding to different domains of the laminin molecule (CSRAKQAASIKVASADR, FALRGDNP, CLQDGDVRV) did not influence type IV collagenase mRNA levels. Zymographic analysis of medium collected from these cultures revealed that neither soluble laminin nor any of the peptides influenced 72-kDa type IV collagenase protein levels. However, peptide FALRGDNP resulted in both, a selective increase in two higher molecular-weight metalloproteinases (83 kDa and 110 kDa and in an activation of the 72-kDa rat type IV collagenase. Interleukin-1, phorbol ester, testosterone, and FSH did not affect collagenase activation. Immunocytochemical studies demonstrated that the addition of soluble laminin resulted in a redistribution of type IV collagenase from intracellular vesicles to the cell-substrate region beneath the cells. Peptide FALRGDNP induced a change from a vesicular to peripheral plasma membrane type of staining pattern. Zymography of plasma membrane preparations demonstrated triton-soluble gelatinases of 76 kDa, 83 kDa, and 110 kDa and a triton-insoluble gelatinase of 225 kDa. These results indicate that testicular cell type IV collagenase mRNA levels, enzyme activation, and distribution are influenced by laminin and RGD-containing peptides.

Amino Acid Sequence↗
PubMed2003

Potassium N-(6-amino-3,4-dihydro-3-methyl-5-nitroso-4-oxopyrimidin-2-yl)-(S)-aspartate N-(6-amino-3,4-dihydro-3-methyl-5-nitroso-4-oxopyrimidin-2-yl)-(S)-aspartic acid 4.88-hydrate: a two-dimensional coordination polymer.

In the title compound, K(+).C(9)H(10)N(5)O(6)(-).C(9)H(11)N(5)O(6).4.88H(2)O, the cations are eight-coordinate. There is a one-dimensional coordination-polymer substructure, built from K and O atoms only, in the form of chains of distorted confacial octahedra, and these are linked into sheets by the neutral organic ligand. The sheets are linked into a single framework by a combination of N-H.O and O-H.O hydrogen bonds.

Journal Article↗
PubMed2000

Electron density and electrostatic properties of two peptide molecules: tyrosyl-glycyl-glycine monohydrate and glycyl-aspartic acid dihydrate.

The electron density and electrostatic properties of Tyr-Gly-Gly and Gly-Asp molecules have been determined from high-resolution X-ray diffraction data at 123 K. Topological properties of the charge distribution are discussed and compared with those derived from other experimental studies on peptide molecules, and the characteristics of the (3,-1) critical points of the C=O, C-N, C-C bonds are analysed. Crystal data for Tyr-Gly-Gly: C13H17N3O5.H2O, Mr = 313, orthorhombic, P2(1)2(1)2(1), Z = 4, T = 123 +/- 2 K; lattice parameters: a = 7.984 (2), b = 9.535 (3), c = 18.352 (5) A, V= 1397.1 (6) A3, Dx = 1.49 g cm(-3), mu = 1.2 cm(-1) for lambdaMo = 0.7107 A. Crystal data for Gly-Asp: C6H10N2O5.2H2O, Mr = 212, orthorhombic, P2(1)2(1)2(1), Z = 4, T = 123 +/- 2 K; lattice parameters: a = 9.659 (1), b = 9.672 (1), c = 10.739 (1) A, V= 1003.3 (4) A3, Dx = 1.40 g cm(-3), mu = 1.3 cm(-1) for lambda(Mo)= 0.7107 A.

Crystallography, X-Ray↗
PubMed1993

N-methyl-D-aspartic acid differentially regulates extracellular dopamine, GABA, and glutamate levels in the dorsolateral neostriatum of the halothane-anesthetized rat: an in vivo microdialysis study.

The effects of local perfusion with the glutamate receptor agonist NMDA and the noncompetitive NMDA receptor antagonist dizolcipine (MK-801) on extracellular dopamine (DA), GABA, and glutamate (Glu) levels in the dorsolateral striatum were monitored using in vivo microdialysis in the halothane-anesthetized rat. In addition, the sensitivity of both the basal and NMDA-induced increases in levels of these neurotransmitter substances to perfusion with tetrodotoxin (TTX: 10(-5) M) and a low Ca2+ concentration (0.1 mM) was studied. The results show that the local perfusion (10 min) with both the 10(-3) and 10(-4) M dose of NMDA increased striatal DA and GABA outflow, whereas only the (10(-3) M) dose of NMDA was associated with a small and delayed increase in extracellular Glu levels. The NMDA-induced effects were dose-dependently counteracted by simultaneous perfusion with MK-801 (10(-6) and 10(-5) M). Both the basal and NMDA (10(-3) M)-induced increase in extracellular striatal DA content was reduced in the presence of TTX and a low Ca2+ concentration, whereas both basal and NMDA-stimulated GABA levels were unaffected by these treatments. Both the basal and NMDA-stimulated Glu levels were enhanced following TTX treatment, whereas perfusion with a low Ca2+ concentration reduced basal Glu levels and enhanced and prolonged the NMDA-induced stimulation. These data support the view that NMDA receptor stimulation plays a role in the regulation of extracellular DA, GABA, and Glu levels in the dorso-lateral neostriatum and provide evidence for a differential effect of NMDA receptor stimulation on these three striatal neurotransmitter systems, possibly reflecting direct and indirect actions mediated via striatal NMDA receptors.

Anesthesia↗
PubMed2001

Variants other than aspartic acid at codon 69 of the human immunodeficiency virus type 1 reverse transcriptase gene affect susceptibility to nuleoside analogs.

The T69D mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) gene has been associated with reduced susceptibility to dideoxycytosine (ddC); however, several other mutations at codon 69 have been observed in antiretroviral drug-treated patients. The Stanford HIV RT and Protease Sequence Database was interrogated and showed that 23% of patients treated with nucleoside RT inhibitors (NRTI) had mutations at codon 69. These variants included T69N, -S, -A, -G, -E, -I, and -K mutations that were present in patients treated with NRTI but not in drug-naive patients. Treatment history information showed that a substantial percentage of these codon 69 changes occurred in patients administered non-ddC-containing regimens. Different and specific patterns of other RT gene mutations were associated with the various codon 69 mutations. Drug susceptibility assays showed that viral constructs containing codon 69 variants could have reduced susceptibility to ddC and other RT inhibitors. These results suggest that the T69D mutation is not the only codon 69 variant associated with drug resistance and that ddC is not the only drug affected.

Codon↗