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S A Carr

Publications and source records attributed to S A Carr.

At least 19 recordsLinked to original sources
PubMed2001

The srhSR gene pair from Staphylococcus aureus: genomic and proteomic approaches to the identification and characterization of gene function.

Systematic analysis of the entire two-component signal transduction system (TCSTS) gene complement of Staphylococcus aureus revealed the presence of a putative TCSTS (designated SrhSR) which shares considerable homology with the ResDE His-Asp phospho-relay pair of Bacillus subtilis. Disruption of the srhSR gene pair resulted in a dramatic reduction in growth of the srhSR mutant, when cultured under anaerobic conditions, and a 3-log attenuation in growth when analyzed in the murine pyelonephritis model. To further understand the role of SrhSR, differential display two-dimensional gel electrophoresis was used to analyze the cell-free extracts derived from the srhSR mutant and the corresponding wild type. Proteins shown to be differentially regulated were identified by mass spectrometry in combination with protein database searching. An srhSR deletion led to changes in the expression of proteins involved in energy metabolism and other metabolic processes including arginine catabolism, xanthine catabolism, and cell morphology. The impaired growth of the mutant under anaerobic conditions and the dramatic changes in proteins involved in energy metabolism shed light on the mechanisms used by S. aureus to grow anaerobically and indicate that the staphylococcal SrhSR system plays an important role in the regulation of energy transduction in response to changes in oxygen availability. The combination of proteomics, bio-informatics, and microbial genetics employed here represents a powerful set of techniques which can be applied to the study of bacterial gene function.

Amino Acid Sequence↗
PubMed2001

Cell cycle regulation of myosin-V by calcium/calmodulin-dependent protein kinase II.

Organelle transport by myosin-V is down-regulated during mitosis, presumably by myosin-V phosphorylation. We used mass spectrometry phosphopeptide mapping to show that the tail of myosin-V was phosphorylated in mitotic Xenopus egg extract on a single serine residue localized in the carboxyl-terminal organelle-binding domain. Phosphorylation resulted in the release of the motor from the organelle. The phosphorylation site matched the consensus sequence of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibitors of CaMKII prevented myosin-V release. The modulation of cargo binding by phosphorylation is likely to represent a general mechanism regulating organelle transport by myosin-V.

Amino Acid Sequence↗
PubMed2001

Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase.

The budding yeast transcriptional activator Gcn4 is rapidly degraded in an SCF(Cdc4)-dependent manner in vivo. Upon fractionation of yeast extracts to identify factors that mediate Gcn4 ubiquitination, we found that Srb10 phosphorylates Gcn4 and thereby marks it for recognition by SCF(Cdc4) ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Gcn4 is almost completely stabilized in srb10Delta pho85Delta cells, or upon mutation of all Srb10 phosphorylation sites within Gcn4, suggesting that the Pho85 and Srb10 cyclin-dependent kinases (CDKs) conspire to limit the accumulation of Gcn4. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat-stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild-type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. We propose that Srb10 also inhibits gene expression by promoting the rapid degradation or nuclear export of specific transcription factors. Simultaneous down-regulation of both transcriptional regulatory proteins and RNA polymerase may enhance the potency and specificity of transcriptional inhibition by Srb10.

Amino Acids↗
PubMed2001

A multidimensional electrospray MS-based approach to phosphopeptide mapping.

A new, multidimensional electrospray MS-based strategy for phosphopeptide mapping is described which eliminates the need to radiolabel protein with 32P or 33P. The approach utilizes two orthogonal MS scanning techniques, both of which are based on the production of phosphopeptide-specific marker ions at m/z 63 and/or 79 in the negative ion mode. These scan methods are combined with liquid chromatography-electrospray mass spectrometry and nanoelectrospray MS/MS to selectively detect and identify phosphopeptides in complex proteolytic digests. Low-abundance, low-stoichiometry phosphorylation sites can be selectively determined in the presence of an excess of nonphosphorylated peptides, even in cases where the signal from the phosphopeptide is indistinguishable from background in the conventional MS scan. The strategy, which has been developed and refined in our laboratory over the past few years, is particularly well suited to phosphoproteins that are phosphorylated to varying degrees of stoichiometry on multiple sites. Sensitivity and selectivity of the method are demonstrated here using model peptides and a commercially available phosphoprotein standard. In addition, the strategy is illustrated by the complete in vitro and in vivo phosphopeptide mapping of Sic1p, a regulator of the G1/S transition in budding yeast.

Amino Acid Sequence↗
PubMed2001

Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry.

Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H3PO4 and HPO3. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta-casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites. This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.

Amino Acid Sequence↗
PubMed1999

Mechanism of inhibition of cathepsin K by potent, selective 1, 5-diacylcarbohydrazides: a new class of mechanism-based inhibitors of thiol proteases.

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.

Binding Sites↗
PubMed1999

Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.

Alkylating Agents↗
PubMed1999

Mammalian homologues of the Drosophila slit protein are ligands of the heparan sulfate proteoglycan glypican-1 in brain.

Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, we have isolated from rat brain at least two proteins that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained by nano-electrospray tandem mass spectrometry. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction and enabled us to isolate from a rat brain cDNA library a 4.1-kilobase clone that encoded two of our peptide sequences and represented the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also showed that our peptides were derived from proteins that are members of the Slit/MEGF protein family, which share a number of structural features such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs, and in Drosophila Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and our peptide sequences correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of the heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using our affinity matrix. Northern analysis demonstrated the presence of two mRNA species of 8. 6 and 7.5 kilobase pairs using probes based on both N- and C-terminal sequences, and in situ hybridization histochemistry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where we have previously also demonstrated glypican-1 mRNA and immunoreactivity. Our results therefore indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis.

Amino Acid Sequence↗
PubMed1999

Isolation and identification of a mouse brain protein recognized by antisera to heart fatty acid-binding protein.

Although a novel brain-specific fatty acid-binding protein (B-FABP) was recently cloned, the identity of a second fatty acid-binding protein detected with antibodies to the heart (H-FABP) has not been clearly resolved. The present investigation, using matrix-assisted laser desorption mass spectrometry, showed that this protein was a form of H-FABP whose N-terminal amino acid was neither methionine nor was it acetylated. Furthermore, isoelectric focusing revealed two major isoforms, a major band pl 7.4 and a minor band pl 6.4, in a distribution pattern opposite to that observed for H-FABP in the heart. Tryptic peptide mass maps of the in-gel digested SDS polyacrylamide gel electrophoresis protein bands showed that the two isoforms differed only in a single peptide corresponding to residues 97-106 of the heart H-FABP sequence. This peptide had an [M + H]+ ion of either 1205.62 (pl 7.4) or 1206.53 (pl 6.4), consistent with a single amino acid substitution, Asp98 or Asn98. Whereas it is well established that both H-FABP and B-FABP interact with polyunsaturated fatty acids, we showed that they also significantly alter plasma membrane cholesterol dynamics in a manner opposite to that of another brain lipid-binding protein, sterol carrier protein-2. In summary, the data demonstrated for the first time that the H-FABP from brain, while nearly identical to H-FABP from heart, differed significantly in isoform distribution and in amino terminal structure from heart H-FABP. This suggests that the brain and heart H-FABP may not necessarily function identically in these tissues.

Amino Acid Sequence↗
PubMed1998

Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis.

Mass spectrometry occupies a central position in most current protein identification schemes. So-called 'mass fingerprinting' techniques rely on composite mass patterns of proteolytic fragments, or dissociation products thereof, to query databases. Keys to successful analysis of ever smaller amounts are sensitivity and complete spectral information, both of which depend for a large part on proper sample preparation. Clean-up and concentration of peptide mixtures over eppendorf gel loading tips filled with chromatographic media (i.e. 'micro-tips') are believed to be quite useful in this regard. We have studied quantitative and qualitative aspects of polypeptide extraction using these small manual devices. Optimization of sample volume and additives, micro-tip bed volume, and eluent composition and volume, all contribute to effective recovery (approximately 65-70%, on average). Improper digest conditions can, in fact, lead to far bigger losses, suggesting the need for at least trace amounts of Zwittergent 3-16. Of particular interest is our finding that partial fractionation, obtained by two-step micro-tip elution, generally results in more and better signals during subsequent mass analysis. Thus, by using optimized micro-tips, in combination with adequate sample handling and instrumentation, direct mass spectrometric identification can be routinely and successfully done in any resource facility type setting.

Amino Acid Sequence↗
PubMed1998

Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry.

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.

Electrophoresis, Capillary↗
PubMed1998

Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior.

The hypothalamus plays a central role in the integrated control of feeding and energy homeostasis. We have identified two novel neuropeptides, both derived from the same precursor by proteolytic processing, that bind and activate two closely related (previously) orphan G protein-coupled receptors. These peptides, termed orexin-A and -B, have no significant structural similarities to known families of regulatory peptides. prepro-orexin mRNA and immunoreactive orexin-A are localized in neurons within and around the lateral and posterior hypothalamus in the adult rat brain. When administered centrally to rats, these peptides stimulate food consumption. prepro-orexin mRNA level is up-regulated upon fasting, suggesting a physiological role for the peptides as mediators in the central feedback mechanism that regulates feeding behavior.

Animals↗
PubMed1998

Isolation and characterization of 26- and 30-kDa rat liver proteins immunoreactive to anti-sterol carrier protein-2 antibodies.

Although the existing literature suggests that the sterol carrier protein-2 (SCP-2) gene has only two initiation sites encoding for a 58- and a 15-kDa protein, respectively, this does not explain the profusion of other putative SCP-2-related proteins detectable on Western blotting. Two of these additional anti-SCP-2 immunoreactive proteins, 13.2 and 46 kDa, appear due to proteolytic processing of the two gene transcripts. However, the origin of additional immunoreactive rat liver proteins near 26 and 30 kDa is unclear. The latter proteins were consistently detected on Western blotting by three independent types of polyclonal antisera: anti-13.2-kDa SCP-2, anti-synthetic peptide from the amino-terminus of the 13.2-kDa SCP-2, and Protein A affinity-purified anti-synthetic peptide to the amino-terminus of 13.2-kDa SCP-2. To resolve whether the 26- and 30-kDa proteins are SCP-2 gene products, each protein was isolated from rat liver and purified to homogeneity as indicated by Tricine-SDS polyacrylamide gel electrophoresis, isoelectric focusing, and/or mass spectroscopy. Their masses, determined by MALDI-TOF mass spectroscopy, were 25.7 and 29.8 kDa, respectively. However, the mass spectral data were not consistent with either protein being an SCP-2 gene product. Peptide mass mapping of the 25.7-kDa protein revealed identity to the rat 25,784.79-Da glutathione-S-transferase. Furthermore, neither the mass nor the amino acid composition of the 29.8-kDa protein correlated with any SCP-2 gene product or dimerized SCP-2 gene product. A database search of the amino acid composition identified the protein as rat carbonic anhydrase. In summary, although the 26- and 29.8-kDa proteins may share some common epitopes with the 13.2-kDa SCP-2, they were not SCP-2 gene products.

Amino Acid Sequence↗
PubMed1997

Design of potent and selective human cathepsin K inhibitors that span the active site.

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Binding Sites↗
PubMed1997

Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase.

G1 cyclin-dependent kinase (Cdk)-triggered degradation of the S-phase Cdk inhibitor Sic1p has been implicated in the transition from G1 to S phase in the cell cycle of budding yeast. A multidimensional electrospray mass spectrometry technique was used to map G1 Cdk phosphorylation sites in Sic1p both in vitro and in vivo. A Sic1p mutant lacking three Cdk phosphorylation sites did not serve as a substrate for Cdc34p-dependent ubiquitination in vitro, was stable in vivo, and blocked DNA replication. Moreover, purified phosphoSic1p was ubiquitinated in cyclin-depleted G1 extract, indicating that a primary function of G1 cyclins is to tag Sic1p for destruction. These data suggest a molecular model of how phosphorylation and proteolysis cooperate to bring about the G1/S transition in budding yeast.

Amino Acid Sequence↗
PubMed1997

Calmodulin modulates the interaction between IQGAP1 and Cdc42. Identification of IQGAP1 by nanoelectrospray tandem mass spectrometry.

Calmodulin regulates numerous fundamental metabolic pathways by binding to and modulating diverse target proteins. In this study, calmodulin-binding proteins were isolated from normal (Hs578Bst) and malignant (MCF-7) human breast cell lines with calmodulin-Sepharose and analyzed by SDS-polyacrylamide gel electrophoresis. A protein that migrated at approximately 190 kDa bound to calmodulin in the presence of Ca2+ and was the only calmodulin-binding protein detected in the absence of Ca2+. This 190-kDa protein was identified as IQGAP1 by nanoelectrospray mass spectrometry and collision-induced dissociation tandem mass spectrometry. IQGAP1 coimmunoprecipitated with calmodulin from lysates of MCF-7 cells. Moreover, overlay with 125I-calmodulin confirmed that IQGAP1 binds directly to calmodulin. Analysis of the functional effects of the interaction revealed that Ca2+/calmodulin disrupted the binding of purified IQGAP1 to the Ras-related protein Cdc42 in a concentration-dependent manner. These data clearly identify IQGAP1 as the predominant calmodulin-binding protein in Ca2+-free breast cell lysates and reveal that calmodulin modulates the interaction between IQGAP1 and Cdc42.

Calmodulin↗
PubMed1997

Autocatalytic activation of human cathepsin K.

The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells. Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were identified as resulting from cleavages after Glu19, Ser98, and Glu110. The mature enzyme was composed of mixture of enzymes with N termini of Gly113, Arg114, and Ala115 with varying ratios depending on the preparation. Molecular weight determinations were consistent with the absence of carbohydrate in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide linkage, and that the protein had Met329 as the C-terminal residue. A mutant was constructed in which the active site Cys139 was changed to Ser. [Ser139,Ala163]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type. [Ser139,Ala163]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.

Amino Acid Sequence↗