The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries four distinct DNA regions in its chromosome, termed pathogenicity islands (PAIs I sub(536) to IV sub(536)). Each of these PAIs ...encodes at least one virulence factor. All four PAIs are associated with tRNA genes. PAI I sub(536) and PAI II sub(536) can be spontaneously deleted from the chromosome by homologous recombination between flanking direct repeats. The deletion of PAI II sub(536) results in the truncation of the associated gene leuX encoding the tRNA sub(5) super(Leu). This tRNA influences the expression of various virulence traits. In order to get a deeper insight into the role of PAI I sub(536)/II sub(536) and of the tRNA sub(5) super(Leu) for the protein expression, the protein expression patterns of Escherichia coli 536 and different derivatives were studied. Differences in the protein expression patterns of the wild-type strain Escherichia coli 536, its mutants 536-21 (PAI I sub(536) super(-), PAI II sub(536) super(-), leuX super(-)), 536 Delta 102 (PAI I sub(536) super(+), PAI II sub(536) super(+), leuX super(-)) as well as of the strain 536R3 (PAI I sub(536) super(-), PAI II sub(536) super(-), leuX super(+)) were analyzed by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry. We identified about 39 different intracellular proteins whose expression is markedly altered in the different strain backgrounds. These differences can be linked either to the presence or absence of the PAI I sub(536) and PAI II sub(536) or to that of the tRNA gene leuX. The identities of 34 proteins have been determined by MALDI-TOF-MS. The identification of five proteins was not possible. The results suggest that proteome analysis is an efficient approach to study differences in global gene expression. The comparison of protein expression patterns of the uropathogenic E. coli strain 536 and different derivatives revealed that in this strain the expression of various proteins including those encoded by many housekeeping genes is affected by the presence of PAI I sub(536) and Pai II sub(536) or by that of the tRNA sub(5) super(Leu).
With the aim to localize the structural region that becomes first accessible to proteolytic attack during thermal unfolding, the proteolysis of ribonuclease A was studied in the temperature range of ...20–65°C. Subtilisin, proteinase K, and elastase proved to be not appropriate as indicators of thermal unfolding, because even the native protein molecule was cleaved by these proteases. In contrast, chymotrypsin, trypsin, and thermolysin attacked ribonuclease A only after its thermal treatment. For thermolysin and trypsin, the first primary cleavage sites of ribonuclease A could be identified by blotting of the electropho‐retic bands, partial N‐terminal sequencing of the fragments and assignment according to their molecular masses. The results were confirmed by the separation of the proteolytic fragments by HPLC and subsequent matrix‐assisted laser desorption ionization mass spectrometry. The first cleavage sites were determined to be Lys31‐Ser32 and Arg33‐Asn34 for trypsin and Asn34‐Leu35 and Thr45‐Phe46 for thermolysin. Hence the structural region from Lys31 to Leu35, together with the adjacent β‐structure containing Thr45‐Phe46, is suggested to represent a labile region of the ribonuclease A molecule, which becomes exposed at thermal denaturation.
The uropathogenic Escherichia coli strain 536 (O6:K15:H31) carries four distinct DNA regions in its chromosome, termed pathogenicity islands (PAIs I536 to IV536). Each of these PAIs encodes at least ...one virulence factor. All four PAIs are associated with tRNA genes. PAI I536 and PAI II536 can be spontaneously deleted from the chromosome by homologous recombination between flanking direct repeats. The deletion of PAI II536 results in the truncation of the associated gene leuX encoding the tRNALeu. This tRNA influences the expression of various virulence traits. In order to get a deeper insight into the role of PAI I536/II536 and of the tRNA5LeU for the protein expression, the protein expression patterns of Escherichia coli 536 and different derivatives were studied. Differences in the protein expression patterns of the wild-type strain Escherichia coli 536, its mutants 536-21 (PAI I536-, PAI II536-, leuX-), 536delta102 (PAI I536+, PAI II536+, leuX-) as well as of the strain 536R3 (PAI I536-, PAI II536-, leuX+) were analyzed by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry. We identified about 39 different intracellular proteins whose expression is markedly altered in the different strain backgrounds. These differences can be linked either to the presence or absence of the PAI I536 and PAI II536 or to that of the tRNA gene leuX. The identities of 34 proteins have been determined by MALDI-TOF-MS. The identification of five proteins was not possible. The results suggest that proteome analysis is an efficient approach to study differences in global gene expression. The comparison of protein expression patterns of the uropathogenic E. coli strain 536 and different derivatives revealed that in this strain the expression of various proteins including those encoded by many housekeeping genes is affected by the presence of PAI I536 and Pai II536 or by that of the tRNA5Leu.
Solid-phase synthesis of peptide-4-nitroanilides Kaspari, A; Schierhorn, A; Schutkowski, M
International journal of peptide and protein research,
November 1996, Letnik:
48, Številka:
5
Journal Article
Recenzirano
A wide variety of Glu/Asp and Gln containing peptide-4-nitroanilides and other chromogenic peptidyl-arylamides could be quickly synthesized by a Fmoc-based solid-phase synthesis strategy employing ...the side-chain carboxyl groups for transient anchoring to the resin. Suitable synthons for this method, Fmoc-Glu-NH-Np and Fmoc-Asp-NH-Np, were prepared using a diphenylphosphinic chloride-mediated coupling reaction. Peptides of the common structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Glu/Asp, Gln) were synthesized and were shown to be substrates for the protease subtilisin Carlsberg (E.C.3.4.21.14a) and for peptidyl-prolyl cis/trans-isomerases (PPIases E.C. 5.2.1.8.). The method was extended to amino acids possessing a side chain missing an anchor for binding to the matrix. We synthesized Suc-Ala-Phe-Pro-Gln-Phe-NH-Np anchoring the dipeptide derivative Fmoc-Glu-Phe-NH-Np with the carboxyl group to Rink amide resin using standard SPPS procedures. Additionally this procedure allowed us the preparation of peptidyl-arylamides, utilizing the commercial available Fmoc-Glu-OAll as building block. A mixture of pentapeptide-4-nitroanilides with the general sequence Ala-Ala-Xaa-Pro-Gln-NH-Np was synthesized. Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the hydrolysis of the peptide mixture by the protease subtilisin Carlsberg. It could be shown that peptides with the hydrophobic amino acids Phe, Tyr, Leu and Val in the varied P3-position were most rapidly cleaved under the chosen conditions. Hydrolysis of the Gln-NH-Np bond in Ala-Ala-Pro-Pro-Gln-NH-Np has not been observed.
In the amino‐acid‐fermenting anaerobe Eubacterium acidaminophilum, acetyl phosphate is synthesized by protein C of glycine reductase from a selenoprotein A‐bound carboxymethyl‐selenoether. We ...investigated specific thiols present in protein C for responsibility for acetyl phosphate liberation. After cloning of the genes encoding the large and the small subunit (grdC1, grdD1), they were expressed separately in Escherichia coli and purified as Strep‐tag proteins. GrdD was the only subunit that catalysed arsenate‐dependent hydrolysis of acetyl phosphate (up to 274 U·mg−1), whereas GrdC was completely inactive. GrdD contained two cysteine residues that were exchanged by site‐directed mutagenesis. The GrdD(C98S) mutant enzyme still catalysed the hydrolysis of acetyl phosphate, but the GrdD(C359A) mutant enzyme was completely inactive. Next, these thiols were analysed further by chemical modification. After iodoacetate treatment of GrdD, the enzyme activity was lost, but in the presence of acetyl phosphate enzyme activity was protected. Subsequently, the inactivated carboxymethylated enzyme and the protected enzyme were both denatured, and the remaining thiols were pyridylethylated. Peptides generated by proteolytic cleavage were separated and subjected to mass spectrometry. Cys98 was not accessible to carboxymethylation by iodoacetate in the native enzyme in the presence or absence of the substrate, but could be alkylated after denaturation. Cys359, in contrast, was protected from carboxymethylation in the presence of acetyl phosphate, but became accessible to pyridylethylation upon prior denaturation of the protein. This clearly confirmed the catalytic role of Cys359 as the active site thiol of GrdD responsible for liberation of acetyl phosphate.
Two series of
N-aminoacyl,
O-benzoyl hydroxamates were designed to investigate the influence of the substituted benzoyl residue on on the hydrolytic stability and the reactivity of these potential ...inhibitors towards selected cysteine and serine proteinases. The inactivators react more rapidly with cysteine proteinases than with the serine enzymes tested. While ZPheGlyNHONbz is the most reactive inhibitor of cathepsin L, inhibiting the target protein by a second order rate constant of 932.000M
−1 s
−1, the bacterial serine proteinase thermitase is inhibited best by ZGlyPheNHONbz, exhibiting a second order rate constant of 1.170 M
−1 s
−1. Thiolsubtilisin, having the thiol-group as the reactive nucleophile instead of serine, exhibits specificity constants of the inactivation two orders of magnitude smaller than subtilisin. The degree of selectivity of the inhibitors relative to cathepsin B, cathepsin L, cathepsin S and papain varies up to two orders of magnitude with respect to their second order rate constant of inactivation. The inhibitory reactivity of these compounds varies only up to sixfold depending on the benzoyl substituent. Similarly, the rate constants for the hydrolytic decomposition of the compounds vary by a factor of about 6, suggesting that the structural and mechanistic features of the compounds which are responsible for decomposition as well as for the enzyme inhibition are the same. Comparing both reactions, the data allow the calculation of an acceleration factor of 2.4 × 10
10 for the inhibition of cathepsin L by its most effective inhibitor, clearly characterizing this enzyme inhibition reaction as enzyme-activated.
The influence of Cd(II), Cu(II) and Zn(II) on growth and thiol production in an aquatic hyphomycete (Articulospora tetracladia) and a zygomycete (Mucor racemosus) were studied. Growth of a strain of ...M. racemosus, isolated from effluents contaminated by heavy metals, was not inhibited by Cd concentrations of up to 100 μM. M. racemosus was more sensitive to Cu and Zn. The production of SH-containing compounds by M. racemosus was positively correlated with Cd concentration in the nutrient medium. By contrast, glutathione concentration in the mycelium was negatively correlated with Cd concentration. Addition of Zn or Cu to the medium had no significant effect on mycelial levels of thiol compounds or glutathione. One strain of A. tetracladia, isolated from a clean stream (At-BB), significantly increased the production of thiol compounds and of glutathione in the presence of Cd; the other strain from a Cu enriched stream (At-CS) showed no significant response. Addition of Zn or Cu did not significantly change levels of thiols or glutathione, with one exception: glutathione levels in At-CS were negatively correlated with external Cu levels. Declining glutathione levels in M. racemosus exposed to Cd was coupled with the appearance of several phytochelatins. No such compounds were found in A. tetracladia.
Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV ...transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP.
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