N-terminal-methionylated interleukin-1 beta has reduced receptor-binding affinity

The receptor-binding affinity of recombinant-derived interleukin-1 beta containing unprocessed N-terminal methionine (MAPV-) was 10-fold lower than protein containing the authentic N-terminal sequence (APV-). Structural analysis of the methionylated and non-methionylated proteins by NMR spectroscopy detected no (or minor) conformational differences. The differences in binding affinity, therefore, suggest that the additional N-terminal methionine causes a small, direct or indirect, perturbation of the receptor-binding region.     

Purification and characterization of a methionine-specific aminopeptidase from Salmonella typhimurium

An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.     

Structure-activity relationship study of human interleukin-3: role of the C-terminal region for biological activity.

A structure-activity relationship study of human interleukin-3 (huIL-3) was performed by functional analysis of huIL-3 deletion and substitution variants combined with epitope mapping of huIL-3 specific neutralizing monoclonal antibodies (mAb). Analysis of the huIL-3 variants was accomplished by defining their capacity to compete with wild-type huIL-3 for binding to the huIL-3 receptor and to induce the proliferation of the huIL-3 dependent cell line M-O7. HuIL-3 variants with either 14 amino acids (aa) deleted from the N-terminus or eight aa from the C-terminus retained full biological activity in vitro. An huIL-3 variant, with 18 N-terminal aa deleted, exhibited a greater than 7-fold reduced receptor binding capacity and proliferative activity. No biological activity could be detected with a variant where 22 C-terminal aa have been deleted. Neutralizing mAb recognizing presumed discontinuous epitopes failed to interact with the latter deletion variant indicating a possible location of their epitopes within the C-terminal region. Computer-aided structure prediction and sequence homology analysis of this region indicated the presence of an amphiphilic alpha-helix with highly conserved residues like Lys110 and Leu111. Substitution of Lys110 with either Glu or Ala resulted in variants with a 10-fold reduced activity in the receptor binding assay and the proliferation assay. Further variants, where Leu111 was substituted by Pro or Met, were totally inactive in these assays. Analysis of the binding of the two neutralizing mAb to these substitution variants showed that they did not bind to either of the Leu111 variants suggesting that Leu111 is part of an active site. Based on our results, a possible model for the structure of the huIL-3 molecule can be constructed with two active sites in close proximity.     

pepM is an essential gene in Salmonella typhimurium.

The pepM gene of Salmonella typhimurium codes for a methionine-specific aminopeptidase that removes N-terminal methionine residues from proteins. This gene was inactivated in vitro by the insertion of a DNA fragment coding for kanamycin resistance. The inactivated gene could not replace the wild-type chromosomal pepM gene unless another functional copy was present in the cell. The lethal effect of the pepM insertion was not a result of polarity on any gene downstream, nor was it affected by the presence or absence of other peptidases.     

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.     

Gene dosage effects of the structural gene for a lipoprotein of the Escherichia coli outer membrane.

The gene dosage effects of the structural gene (lpp) for the lipoprotein of the Escherichia coli outer membrane were examined. A novel F-prime factor containing the lpp gene was constructed. The amount of the free-form lipoprotein in the merodiploid strain carrying the F-prime factor was found to be about two times as great as that in the corresponding haploid strain. On the other hand, the amount of the bound-form lipoprotein, which is vovalently linked to the peptidoglycan, was not significantly different in the merodiploid strain as compared with the corresponding haploid strain. The present results suggest that the lpp gene is expressed constitutively in contrast to another major protein of the E. coli outer membrane, tolG protein (protein II, D. B. Datta et al., J. Bacteriol. 128:834-841, 1976). The F-prime factor isolated may include a portion of the E. coli chromosome (located between 33 and 36 min on the genetic map) that is not covered by any other F-prime factor.     

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin

1. Download : Download high-res image (307KB)
2. Download : Download full-size image

Highlights? The fungal secondary metabolite Cladosporin inhibits liver- and blood-stage malaria parasites ? Cladosporin specifically targets lysyl-tRNA synthetase (Krs1) ? Cladosporin is >100-fold more potent against parasite Krs1 relative to the human enzyme ? Two amino acids in the Krs1 ATP-binding pocket confer species-selective inhibition     

Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients

Patients with coronary heart disease or equivalent risk received a single dose of 30, 100, 300, or 500 mg of unformulated D-4F (n = 8, each dose) or placebo (n = 8) under fasting conditions. An additional 10 patients received 500 mg (n = 8) or placebo (n = 2) with a low-fat meal. There were no significant trends in any safety parameter. D-4F was detectable in plasma at all doses with a T(max) of 30 min, 1 h, and 2 h for 30, 100, and > or = 300 mg, respectively. The area under the curve((0-t)) was 27.81 ng/hr/ml and 54.71 ng/hr/ml for the 300 mg and 500 mg dose groups, respectively, and 17.96 ng/hr/ml for the 500 mg dose given with food. HDL from each time point for each subject was tested for its ability to inhibit LDL-induced monocyte chemotactic activity in cultures of human aortic endothelial cells. The values obtained were normalized to 1.0 for LDL alone to obtain the HDL inflammatory index. This index significantly improved at 4 h at the 300 mg dose and at 2 h at the 500 mg dose compared with placebo (P < 0.05). There were no changes in plasma lipid or lipoprotein levels. We conclude that unformulated D-4F has low bioavailability that is improved under fasting conditions, and that a single dose of D-4F is safe and well tolerated and may improve the HDL anti-inflammatory index.