Jiri Jiracek

Betaine-homocysteine methyltransferase: zinc in a distorted barrel.

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.

Changes in the proteomes of the hemocytes and fat bodies of the flesh fly Sarcophaga bullata larvae after infection by Escherichia coli

BackgroundInsects have an efficient self-defense system that is based on innate immunity. Recent findings have disclosed many parallels between human and insect innate immunity, and simultaneously fine differences in the processes between various species have been revealed. Studies on the immune systems of various insect species may uncover the differences in their host defense strategies.ResultsWe analyzed the proteomes of the hemocytes and fat bodies of Sarcophaga bullata larvae after infection by Escherichia coli. The 2-DE gels of the hemocytes and fat bodies of infected larvae were compared with those of aseptically injured larvae. Our analysis included the construction of protein maps of the hemocyte cells and cells from fat bodies, the identification of the changed proteins, in response to infection, using LC-MS/MS, and the estimation of the trends in expression of these proteins at three time points (30 min, 6 hours and 22 hours) after infection. In total, seven changed spots were found in the hemocytes, and four changed spots were found in the fat bodies. Three types of trends in protein expression were observed. Cofilin and transgelin were undetectable at 30 min after infection but were continuously up-regulated in the induced larvae after 22 hours. A prophenoloxidase isoform and lectin subunit α were slightly up-regulated at 30 min after infection, and their protein levels reached the highest points after 6 hours but decreased after 22 hours. T-Complex subunit α, GST, ferritin-like protein and an anterior fat body protein (regucalcin homologue) were down-regulated at 22 hours after infection.ConclusionsMany proteins identified in our study corresponded to the proteins identified in other insects. Compared to the former studies performed in insects, we presented 2-D protein maps of the hemocytes and fat bodies and showed the trends in expression of the immune-elicited proteins.

Examination of the role of endopeptidase 3.4.24.15 in Aβ secretion by human transfected cells

We have taken advantage of our recent development of highly potent and specific phosphinic inhibitors of endopeptidase 3.4.24.15 to examine the putative contribution of the enzyme in the secretion of Aβ by HK293 transfected cells overexpressing the wild type and the Swedish (Sw) double mutated form of βAPP751. First, we showed that HK293 cells contain a peptidase activity, the inhibition profile of which fully matches that of purified endopeptidase 3.4.24.15. Second, we established that the treatment of HK293 cells with specific phosphinic inhibitors leads to about 80% inhibition of intracellular endopeptidase 3.4.24.15 activity, indicating that these inhibitors penetrate the cells. Metabolic labelling of wild type and SwβAPP751‐expressing cells, followed by immunoprecipitation of Aβ‐containing peptides, revealed the secretion of Aβ and the intracellular formation of an Aβ‐containing 12 kDa product. Aβ secretion by SwβAPP751 transfected cells was drastically enhanced when compared to cells expressing wild type βAPP751. This production was not affected by endopeptidase 3.4.24.15 inhibitors in either cell type. This correlates well with the observation that endopeptidase 3.4.24.15 does not cleave recombinant baculoviral SwβAPP751, in vitro. Our previous data indicated that endopeptidase 3.4.24.15 activity was reduced in the parietal cortex of Alzheimers disease affected brains and that the enzyme probably participated, in this brain area, to the catabolism of somatostatin 1‐14. However, the present work indicates that endopeptidase 3.4.24.15 does not seem to behave as a β‐secretase in HK293 transfected cells. Therefore, it is suggested that endopeptidase 3.4.24.15 could participate in the symptomatology, but probably not in the aetiology of Alzheimers disease.

Dietary intake of S-(α-carboxybutyl)-DL-homocysteine induces hyperhomocysteinemia in rats

Betaine homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to homocysteine (Hcy), forming dimethylglycine and methionine. We previously showed that inhibiting BHMT in mice by intraperitoneal injection of S-(alpha-carboxybutyl)-DL-homocysteine (CBHcy) results in hyperhomocysteinemia. In the present study, CBHcy was fed to rats to determine whether it could be absorbed and cause hyperhomocysteinemia as observed in the intraperitoneal administration of the compound in mice. We hypothesized that dietary administered CBHcy will be absorbed and will result in the inhibition of BHMT and cause hyperhomocysteinemia. Rats were meal-fed every 8 hours an L-amino acid-defined diet either containing or devoid of CBHcy (5 mg per meal) for 3 days. The treatment decreased liver BHMT activity by 90% and had no effect on methionine synthase, methylenetetrahydrofolate reductase, phosphatidylethanolamine N-methyltransferase, and CTP:phosphocholine cytidylyltransferase activities. In contrast, cystathionine beta-synthase activity and immunodetectable protein decreased (56% and 26%, respectively) and glycine N-methyltransferase activity increased (52%) in CBHcy-treated rats. Liver S-adenosylmethionine levels decreased by 25% in CBHcy-treated rats, and S-adenosylhomocysteine levels did not change. Furthermore, plasma choline decreased (22%) and plasma betaine increased (15-fold) in CBHcy-treated rats. The treatment had no effect on global DNA and CpG island methylation, liver histology, and plasma markers of liver damage. We conclude that CBHcy-mediated BHMT inhibition causes an elevation in total plasma Hcy that is not normalized by the folate-dependent conversion of Hcy to methionine. Furthermore, metabolic changes caused by BHMT inhibition affect cystathionine beta-synthase and glycine N-methyltransferase activities, which further deteriorate plasma Hcy levels.

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors.

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.

Evaluation of carrier ampholyte-based capillary electrophoresis for separation of peptides and peptide mimetics

Carrier ampholyte‐based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4–9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline‐rich di‐ to decapeptides, and phosphinic pseudopeptides – tetrapeptide mimetics synthesized as a mixture of four diastereomers having the –P(O)(OH)–CH2– moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.

Synthesis of N-succinyl-L,L-diaminopimelic acid mimetics via selective protection.

The search for potential inhibitors that target so far unexplored bacterial enzyme mono-N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) has stimulated a development of methodology for quick and efficient preparation of mono-N-acylated 2,6-diaminopimelic acid (DAP) derivatives bearing the different carboxyl groups or lipophilic moieties on their amino group.