Changes in fibrinogen and fibrin induced by a peptide analog of fibrinogen gamma365-380.

BACKGROUND: The effects of synthetic peptides with sequences derived from the gamma-chain of fibrinogen on the functional properties of fibrinogen and fibrin were investigated. METHODS: Methods included thrombelastography, clot turbidity measurement, clot elasticity measurement, platelet aggregation, and scanning transmission electron microscopy (STEM). RESULTS: Peptide gamma369-380 (NH(2)-WATWKTRWYSMK-COOH) showed the greatest impact on fibrin structure, compared with the 76 other overlapping dodecapeptides. Addition of this peptide, or peptide gamma365-380 (NH(2)-NGIIWATKTREWYSMK-COOH) to a mixture of fibrinogen and thrombin resulted a shorter clotting time, higher clot turbidity, lower clot elastic modulus, a higher degree of D-trimer and D-tetramer formation, and impaired plasmin proteolysis of the clot. In STEM, fibrin formed in the presence of peptide gamma369-380 consisted of a more extensive array of linear fibrils typically consisting of 20 or more molecules. Fibrils were better organized than those from non-peptide containing mixtures. CONCLUSIONS: Replacement of the tryptophan residue gamma376 massively reduced the effect of the peptide on fibrin structure. Binding of the peptide to fibrinogen induces conformational changes, which result in accelerated clotting and increased lateral association of fibrin protofibrils. The results imply a relevant functional role of sites interacting with peptide gamma369-380 region in the fibrinogen molecule.

Cell fingerprinting: an approach to classifying cells according to mass profiles of digests of protein extracts.

We present a statistical framework for classifying cells according to the set of peptide masses obtained by mass spectrometric analysis of digestions of whole cell protein extracts. The digest is separated by high performance liquid chromatography (HPLC) coupled directly to a mass spectrometer either by an electrospray interface or by collection to a matrix-assisted laser desorption/ionization target plate. Here, the mass to charge ratio, intensity, and HPLC retention time of the peptides are measured. We have used defined bacterial strains to test this approach. For each bacterium, this process is repeated for extracts obtained at different points in the growth curve in order to try and define an invariant set of signals that uniquely identify the bacterium. This paper presents algorithms for the creation of this cell fingerprint database and develops a Bayesian classification scheme for deciding whether or not an unknown bacterium has a match in the database. Our initial testing based on a limited data set of three bacteria indicates that our approach is feasible. Via a jack-knife test, our Bayesian classification scheme correctly identified the bacterium in 67.8% of the cases.

Studies on single-strand scissions to cell-free plasmid DNA by the dopaminergic neurotoxin 'TaClo' (1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline).

The DNA-damaging effect of the potent dopaminergic neurotoxin 'TaClo' (1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline) is reported. After irradiation (300 nm, 350 nm) of cell-free pBR322 DNA in the presence of TaClo, single-strand breaks were observed by gel electrophoresis. It seems likely that TaClo undergoes a light-induced formation of reactive species (probably radicals). In comparison with TaClo, the bromo derivative exhibits an increased toxic action while the fluoro derivative shows a weaker effect towards DNA. In the presence of Cu(II) at 37 degrees C in the dark, DNA strand scissions were found to occur, too. The formation of Cu(I), as detected by UV-absorption at 480 nm using the Cu(I) complexing ligand BCS, hints at the formation of a reactive species by a Cu(II)/Cu(I) mediated redox cycle. Until now, the chemical nature of this reactive species has not been elucidated.

Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety.

We describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one- and two-dimensional polyacrylamide gel electrophoresis. The approach is based on the use of an isotopically labeled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. The method allows quantitation of the changes occurring in spots or bands that contain more than one protein and has a greater dynamic range than most staining methods. Since the reagent carries a fixed positive charge under acidic conditions and labels only the N-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. The sequences can easily be extracted for homology searches instead of using indirect mass spectral-based searches and are independent of posttranslational modifications.

Breakdown of cytoskeletal proteins during meiosis of starfish oocytes and proteolysis induced by calpain.

Meiosis reinitiation in starfish oocytes is characterized by Ca(2+) transients in the cytosol and in the nucleus and is accompanied by the disassembly of the nuclear envelope, a process which is likely to be mediated by the cleavage of selected proteins. We have used mass spectrometry analysis (mass profile fingerprinting) on 2D polyacrylamide gels of extracts of oocytes in which meiosis resumption was induced by 1-methyladenine and have identified five proteins that were specifically degraded: alpha-tubulin, lamin B, dynamin, and two kinds of actin. They are all components of the cytoskeleton or associated with it. We then investigated whether calpain, which is activated by the increase in cell Ca(2+), could cleave the same proteins that became degraded under the influence of 1-methyladenine and thus be involved in nuclear membrane breakdown. The investigation was prompted by the finding that microinjection of calpain into the nuclei of prophase arrested oocytes induced meiosis in the absence of 1-methyladenine. Incubation of prophase arrested (disrupted) oocytes with calpain produced a 2D gel protein pattern in which some of the degradation products coincided with those seen in oocytes challenged with 1-methyladenine.

Differential degradation of Escherichia coli sigma32 and Bradyrhizobium japonicum RpoH factors by the FtsH protease.

The Escherichia coli heat shock sigma factor sigma32 (RpoH) is rapidly degraded under non-stress conditions. The integrity of the DnaK chaperone machinery and the ATP-dependent FtsH protease are required for sigma32 proteolysis in vivo. Bradyrhizobium japonicum expresses three sigma32-type transcription factors, RpoH1, RpoH2, and RpoH3, which are functional in E. coli. We compared the stability of these sigma factors with E. coli sigma32 stability. In E. coli C600 (wild-type), the half-lives of sigma32, RpoH1, RpoH2 and RpoH3 were 30 s, 7 min, 4 min and 4 min, respectively. The first three proteins were stabilized in ftsH mutant backgrounds, indicating that they are degraded by FtsH in the wild-type. Proteolysis of RpoH3 was FtsH-independent because this sigma factor was not stabilized in ftsH mutants. Interestingly, in a purified in vitro system, all four RpoH proteins were degraded by FtsH, indicating that in vivo protein degradation depends on additional cellular factors. Rationally designed point mutations of sigma32 and RpoH1 suggested that the highly conserved RpoH box does not play a major role in conferring stability to RpoH factors. Presumably, several regions distributed along the primary sequence of the sigma factor are important for FtsH-mediated proteolysis. Finally, we provide evidence that proteolysis of RpoH factors in vivo depends on the DnaK machinery, irrespective of the protease involved.

Anthraquinones and betaenone derivatives from the sponge-associated fungus Microsphaeropsis species: novel inhibitors of protein kinases.

An undescribed fungus of the genus Microsphaeropsis, isolated from the Mediterranean sponge Aplysina aerophoba, produces two new betaenone derivatives (1, 2) and three new 1,3,6, 8-tetrahydroxyanthraquinone congeners (5-7). The structures of the compounds were established on the basis of NMR spectroscopic and mass spectrometric data and by CD spectroscopy. This is the first report wherein the (1)H and (13)C NMR data of the betaenone congeners are fully and unambiguously assigned on the basis of two-dimensional NMR spectroscopy. Furthermore, we describe the first elucidation of the absolute configuration of 1-(2'-anthraquinonyl)ethanols such as 5 and 6, by quantum chemical calculation of their circular dichroism (CD) and comparison with experimentally measured spectra. Moreover, it was shown that compounds 1, 5, 6, and 7 are inhibitors of PKC-epsilon, CDK4, and EGF receptor tyrosine kinases.

A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification.

We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database. The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing. The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples. We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins. We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis.

Proteome analysis of heat shock protein expression in Bradyrhizobium japonicum.

A set of 19 heat shock proteins (Hsp) was observed - by subtractive two-dimensional gel electrophoresis - to be induced when Bradyrhizobium japonicum, the nitrogen-fixing root-nodule symbiont of soybean, was temperature up-shifted from 28 degrees C to 43 degrees C. Up-regulated protein spots were excised from multiple two-dimensional gels. The proteins were concentrated using a funnel-gel device before being blotted onto poly(vinylidene difluoride) membranes for digestion with trypsin before MS and tandem MS analysis or for Edman sequence determination. Five proteins in the range 8-20 kDa were identified as the small Hsp (sHsp; HspB, C, D, E and H) and three others showed strong sequence similarity to the sHsp family. Two other low molecular mass proteins corresponded to GroES1 and GroES2, and five novel proteins were found. Four proteins of approximately 60 kDa were identified as GroEL2, GroEL4, and GroEL5 and DnaK. An analysis of the heat shock induction of DnaK, of four of the most strongly induced GroESL proteins and six of the sHsp revealed that the proteins could be placed into four distinct regulatory groups based on the kinetics of protein appearance.

Multiple small heat shock proteins in rhizobia.

Seven genes coding for small heat shock proteins (sHsps) in Bradyrhizobium japonicum have been identified. They are organized in five operons that are coordinately regulated by ROSE, a negatively cis-acting DNA element. The deduced sHsps can be divided into two separate classes: class A, consisting of proteins that show similarity to Escherichia coli IbpA and IbpB, and class B, whose members display significant similarity to other sHsps from prokaryotes and eukaryotes. Two-dimensional gel electrophoresis and Edman sequencing revealed the presence of at least 12 sHsps in B. japonicum, indicating a remarkable abundance of sHsps in this organism. Three additional members of class A and two potentially novel heat shock proteins were identified on the basis of their amino termini. The presence of multiple sHsps was also demonstrated for a variety of Rhizobium and Bradyrhizobium species by immunoblot analysis and two-dimensional gel electrophoresis. An extensive database survey revealed that, in contrast to the rhizobia, other bacteria contain maximally two sHsps whereas many plants have been reported to possess a sHsp superfamily.