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At least 199 records · Page 11Linked to original sources
PubMed1997

Cysteine-rich regions of protein kinase Cdelta are functionally non-equivalent. Differences between cysteine-rich regions of non-calcium-dependent protein kinase Cdelta and calcium-dependent protein kinase Cgamma.

Regulatory domain elements of the non-calcium-dependent protein kinase Cdelta (nPKCdelta), including either or both of the cysteine-rich regions Cys1(delta) and Cys2(delta), were expressed as fusion proteins with glutathione-S-transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(delta) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(delta). Likewise, in mixed micellar assays, fusion proteins with Cys2(delta) bound PDBu with high affinity (Kd: 14-37 nM) and to significant stoichiometric levels (0.23-0.66 mol/mol), but no binding could be detected for proteins with Cys1(delta) only. The PS dependence of PDBu binding to Cys2(delta) was highly cooperative with Hill numbers lying in the range of 2.5-5.2. These results demonstrate the presence of striking functional differences between the cysteine-rich regions of nPKCdelta and the calcium-dependent isoform, cPKCgamma, where both cysteine-rich regions represent functional PDBu binding elements.

Binding Sites↗
PubMed1999

CA-MMP: a matrix metalloproteinase with a novel cysteine array, but without the classic cysteine switch.

A matrix metalloproteinase (MMP)-like gene was identified in mouse to contain a conserved MMP catalytic domain and an RRRR motif. It lacks a classic cysteine switch, but it possesses two novel motifs: a cysteine array (Cys-X(6)-Cys-X(8)-Cys-X(10)-Cys-X(3)-Cys-X(2)-Cys), and a novel Ig-fold. It is named CA-MMP after the distinct cysteine array motif, and little is known about its biochemical function. In an attempt to characterize CA-MMP activity, the full-length sequence was expressed in mammalian cells and its product found to be cell-associated without detectable secretion. In light of this unusual finding, a chimera combining the catalytic domain of CA-MMP with the prodomain of stromelysin-3 was constructed to express a fully active enzyme in mammalian cells. Purified CA-MMP catalytic domain expresses proteolytic activity against protein substrates in an MMP inhibitor sensitive fashion. Taken together, it is concluded that CA-MMP is an MMP with distinct structure, biochemical properties and evolutionary history that may define a new subclass of the MMP superfamily.

Amino Acid Sequence↗
PubMed2001

Expression of cysteine proteinases cathepsins B and K and of cysteine proteinase inhibitor cystatin C in giant cell tumor of tendon sheath.

The expression of cysteine proteinases cathepsins B and K and of the endogenous inhibitor of cysteine proteinases, cystatin C, was investigated in tissue specimens of patients with giant cell tumor of tendon sheath (GCTTS). Expression of both enzymes was examined by immunohistochemistry in tissue specimens of 14 patients with GCTTS. Applying double-labeling techniques, the coexpression of cathepsin B and its major endogenous inhibitor cystatin C was additionally studied. Cells expressing the respective proteins were further characterized with the macrophage markers HAM56 and anti-CD68 (clone PG-M1). Cathepsin B could be detected in numerous HAM56-positive mononuclear cells (MC), but only in very few giant cells (GC). In contrast, cathepsin K was predominantly identified in GC that were also strongly immunoreactive for cystatin C and CD68. Coexpression of cathepsin B and cystatin C occurred only in a few MC. The strong expression of both cathepsin B and K suggests that in GCTTS, bone erosion might be mediated not only by pressure of the proliferative tissue, but also by matrix-degrading cysteine proteinases. Because previous studies showed that osteoclasts express high levels of CD68, cathepsin K, and cystatin C but not of cathepsin B, our study contributes to the view that GC of GCTTS and osteoclasts are closely associated.

Adult↗
PubMed1970

The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.

Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.

Acetone↗
PubMed1991

Effects of site-directed mutagenesis at residues cysteine-31 and cysteine-184 on lecithin-cholesterol acyltransferase activity.

Native lecithin-cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase; phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) protein, and LCAT in which either or both of the enzyme free cysteines had been replaced with glycine residues by site-directed mutagenesis, has been expressed in cultured Chinese hamster ovary cells stably transfected with the human LCAT gene. The mass of LCAT secreted, determined by immunoassay, did not differ in the native and mutant species. LCAT specific activity was also unchanged in the mutant species. In particular, the cysteine-free double mutant, in which Cys-31 and Cys-184 had both been replaced, was fully active in the synthesis of cholesteryl esters. This result is not consistent with a catalytic role for LCAT free cysteine residues. The classical inhibitor of LCAT activity, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which strongly (89%) inhibited the native enzyme, had partial (45%) inhibitory activity with mutant enzyme species containing a single -SH residue, while the double mutant was not significantly inhibited by DTNB. These data are interpreted to suggest that Cys-31 and Cys-184 are vicinal both to each other and to the "interfacial binding site" at residues 177-182, and that DTNB exerts its effect by steric inhibition.

Amino Acid Sequence↗
PubMed2002

Inhibition of plasmodium falciparum triose-phosphate isomerase by chemical modification of an interface cysteine. Electrospray ionization mass spectrometric analysis of differential cysteine reactivities.

Plasmodium falciparum triose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 > Cys-13 Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed.

Amino Acid Sequence↗
PubMed2003

Analysis of transmembrane segment 8 of the GLUT1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility.

The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative alpha-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters.

4-Chloromercuribenzenesulfonate↗
PubMed2004

Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis.

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.

Alanine↗
PubMed2004

Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine.

Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides. The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 microM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (approximately 7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells.

Adenosine Triphosphate↗
PubMed2004

Crystal structure of the cysteine-rich secretory protein stecrisp reveals that the cysteine-rich domain has a K+ channel inhibitor-like fold.

Stecrisp from Trimeresurus stejnegeri snake venom belongs to a family of cysteine-rich secretory proteins (CRISP) that have various functions related to sperm-egg fusion, innate host defense, and the blockage of ion channels. Here we present the crystal structure of stecrisp refined to 1.6-angstrom resolution. It shows that stecrisp contains three regions, namely a PR-1 (pathogenesis-related proteins of group1) domain, a hinge, and a cysteine-rich domain (CRD). A conformation of solvent-exposed and -conserved residues (His60, Glu75, Glu96, and His115) in the PR-1 domain similar to that of their counterparts in homologous structures suggests they may share some molecular mechanism. Three flexible loops of hypervariable sequence surrounding the possible substrate binding site in the PR-1 domain show an evident difference in homologous structures, implying that a great diversity of species- and substrate-specific interactions may be involved in recognition and catalysis. The hinge is fixed by two crossed disulfide bonds formed by four of ten characteristic cysteines in the carboxyl-terminal region and is important for stabilizing the N-terminal PR-1 domain. Spatially separated from the PR-1 domain, CRD possesses a similar fold with two K+ channel inhibitors (Bgk and Shk). Several candidates for the possible functional sites of ion channel blocking are located in a solvent-exposed loop in the CRD. The structure of stecrisp will provide a prototypic architecture for a structural and functional exploration of the diverse members of the CRISP family.

Amino Acid Sequence↗
PubMed2006

Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin.

Among many proteins with cysteine sulfinic acid (Cys-SO2H) residues, the sulfinic forms of certain peroxiredoxins (Prxs) are selectively reduced by sulfiredoxin (Srx) in the presence of ATP. All Srx enzymes contain a conserved cysteine residue. To elucidate the mechanism of the Srx-catalyzed reaction, we generated various mutants of Srx and examined their interaction with PrxI, their ATPase activity, and their ability to reduce sulfinic PrxI. Our results suggest that three surface-exposed amino acid residues, corresponding to Arg50, Asp57, and Asp79 of rat Srx, are critical for substrate recognition. The presence of the sulfinic form (but not the reduced form) of PrxI induces the conserved cysteine of Srx to take the gamma-phosphate of ATP and then immediately transfers the phosphate to the sulfinic moiety of PrxI to generate a sulfinic acid phosphoryl ester (Prx-Cys-S(=O)OPO3(2-)). This ester is reductively cleaved by a thiol molecule (RSH) such as GSH, thioredoxin, and dithiothreitol to produce a disulfide-S-monoxide (Prx-Cys-S(=O)-S-R). The disulfide-S-monoxide is further reduced through the oxidation of three thiol equivalents to complete the catalytic cycle and regenerate Prx-Cys-SH.

Adenosine Diphosphate↗
PubMed1998

Methionine and cysteine kinetics at different intakes of methionine and cysteine in elderly men and women.

Earlier nitrogen balance studies led to the conclusion that requirements for methionine in older individuals are much higher than those in younger adults. Hence, we examined the kinetics of whole-body methionine, cysteine, and leucine metabolism postabsorptively using a continuous intravenous infusion of L-[C2H3, 1-(13)C]methionine, L-[2H3]leucine, and [3,3-2H2]cysteine in 12 elderly men (n = 5) and women (n = 7) given as a 3-h infusion after a 12-h fast (study 1) and in 8 elderly men (n = 4) and women (n = 4) as an 8-h infusion according to a 3-h fasted, 5-h fed protocol (study 2) for 6 d. Before tracer infusion, each of 3 L-amino acid diets supplying the following nominal, but known, amounts (mg x kg(-1) x d(-1)) of methionine and cysteine, respectively, were used in study 2: diet 1: 13 and 0; diet 3: 6.5 and 5.2; and diet 5: 6.5 and 21. Studies 1 and 2 gave values for plasma methionine flux that agreed with the leucine flux data, which, in turn, also appeared to be comparable with findings in healthy younger adults. In study 2, methionine oxidation rates were the same across all diets in the fasted state and the same with diets 1 and 3 in the fed state but lower with diet 5, suggesting a modest sparing effect of dietary cystine on methionine oxidation. Estimated daily methionine balance was at equilibrium for diet 1 and negative (significantly different from zero, P<0.05) with diets 3 and 5. The results were evaluated against our previous findings in younger adults.

Age Factors↗
PubMed1996

Rat liver fatty acid-binding protein: identification of a molecular species having a mixed disulfide with cysteine at cysteine-69 and enhanced protease susceptibility.

Fatty acid-binding protein (FABP) has been isolated from rat liver cytosol by two steps of gel-permeation chromatography on Sephadex G-75 and Sephacryl S-100 after ammonium sulfate precipitation. FABP fraction was eluted as two well-separated peaks, fractions A and B, by reversed-phase high-performance liquid chromatography (HPLC). The structural difference between the two fractions was investigated by lysyl endopeptidase digestion followed by reversed-phase HPLC of the digests, which identified a peptide corresponding to residues 58 through 78 as the modified peptide. Matrix-assisted laser-desorption-ionization mass spectrometry and other chemical analyses of the peptides established the modification in fraction A as cystein-thiolation at cysteine-69. This was confirmed by reduction and reoxidation of the peptide and the parent molecules. The modification did not affect binding of fluorescent derivatives of fatty acids. However, the modified species was more susceptible to proteolysis by bovine spleen cathepsin B and cathepsin D than the unmodified species. The presence of a relatively large amount of cysteine (but not of glutathione) mixed-disulfide form of FABP suggests some physiological role of this modification related to the redox status of the cell [Thomas, J.A., Poland, B., and Honzatko, R. (1995) Arch. Biochem. Biophys. 319, 1-9], and accounts, at least in part, for the extensive heterogeneity of liver FABP.

Amino Acids↗
PubMed1987

L-cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant.

Serine acetyltransferase (SAT) from Escherichia coli is subject to feedback inhibition by L-cysteine. A mutant was isolated which excretes L-cysteine because of a lesion in cysE, the structural gene for SAT, rendering the enzyme less feedback sensitive. To analyse the structural basis for this mutation the cysE genes both from wild-type E. coli and the mutant strain were cloned and their nucleotide sequences determined. The cysE gene contained an open reading frame consisting of 819 bp, equivalent to a protein of 273 amino acids. The mutant gene showed a single base change in position 767 resulting in a methionine to isoleucine substitution. A causal connection between this SAT sequence alteration, feedback insensitivity and L-cysteine excretion was demonstrated. The SAT from the wild-type strain was purified. It was composed of a single polypeptide chain migrating in SDS gels according to an Mr of 34,000. As in Salmonella typhimurium, the enzyme was associated in a bifunctional complex with O-acetylserine (thiol)-lyase.

Acetyltransferases↗
PubMed2002

Prediction of the disulfide-bonding state of cysteines in proteins at 88% accuracy.

The task of predicting the cysteine-bonding state in proteins starting from the residue chain is addressed by implementing a new hybrid system that combines a neural network and a hidden Markov model (hidden neural network). Training is performed using 4136 cysteine-containing segments extracted from 969 nonhomologous proteins of well-resolved three-dimensional structure. After a 20-fold cross-validation procedure, the efficiency of the prediction scores as high as 88% and 84%, when measured on cysteine and protein basis, respectively. These results outperform previously described methods for the same task.

Cysteine↗
PubMed1982

Cysteine sulfinic acid in the central nervous system: specific binding of [35S]cysteic acid to cortical synaptic membranes--an investigation of possible binding sites for cysteine sulfinic acid.

Specific binding sites for cysteine sulfinic acid, an excitatory amino acid, in crude synaptic membrane fractions of rat cerebral cortex were examined, using L-[35S]cysteic acid as a ligand. Two specific binding systems of [35S]cysteic acid were found, one Na+-dependent and the other Na+-independent. The Na+-independent specific binding of [35S]cysteic acid was saturable, with a Kd of 474 nM and Bmax of 3.29 pmol/mg protein. The binding was optimal at pH 7.4 and at 37 degrees C. Treatment of the membranes with proteases, concanavalin A, or Triton X-100 markedly reduced the binding. Of various compounds related to cysteic acid, L-cysteine sulfinic acid was the most effective competitor of this binding. These results indicate the existence of an Na+-independent specific binding site for cysteic acid in the synaptic membrane of rat cerebral cortex, which may be different from that for glutamate. Possible involvement of cysteine sulfinic acid as an endogenous ligand for this binding site is discussed.

Amino Acids, Sulfur↗
PubMed1975

Cysteine biosynthesis in Salmonella typhimurium: the presence of ATP-sulfurylase and APS-kinase in various cysteine-requiring mutants.

Enzymatic tests were performed on a series of cysteine-requiring mutants for the presence of the sulfate activating enzymes. ATP-sulfurylase (sulfate adenylyltransferase EC 2.7.7.4) and APS-kinase (adenylylsulfate kinase EC 2.7.1.25). The enzymatic products adenosine 5'-[35S]sulfatophosphate and adenosine 3'-phosphate 5'-[35S]sulfatophosphate were identified by paper electrophoresis and measured quantitatively without elution from the paper. Cys mutants mapping in cistrons, A, H, I, J, G, and Ea contain both enzymes. Mutation in the D cistron leads to the loss of ATP-sulfurylase. Mutants mapping in the C cistron lack APS-kinase. Ba, Bb, and Bc mutants lack both enzymes. The control of the synthesis of these enzymes by cysteine was examined. Both enzymes are missing when cells are grown on cysteine.

Adenosine Triphosphate↗