Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.</p

Changes in cerebral oxygenation and cerebral blood flow during hemodialysis - A simultaneous near-infrared spectroscopy and positron emission tomography study

Near-infrared spectroscopy (NIRS) is used to monitor cerebral tissue oxygenation (rSO2) depending on cerebral blood flow (CBF), cerebral blood volume and blood oxygen content. We explored whether NIRS might be a more easy applicable proxy to [15O]H2O positron emission tomography (PET) for detecting CBF changes during hemodialysis. Furthermore, we compared potential determinants of rSO2 and CBF. In 12 patients aged ≥ 65 years, NIRS and PET were performed simultaneously: before (T1), early after start (T2), and at the end of hemodialysis (T3). Between T1 and T3, the relative change in frontal rSO2 (ΔrSO2) was -8 ± 9% ( P = 0.001) and -5 ± 11% ( P = 0.08), whereas the relative change in frontal gray matter CBF (ΔCBF) was -11 ± 18% ( P = 0.009) and -12 ± 16% ( P = 0.007) for the left and right hemisphere, respectively. ΔrSO2 and ΔCBF were weakly correlated for the left (ρ 0.31, P = 0.4), and moderately correlated for the right (ρ 0.69, P = 0.03) hemisphere. The Bland-Altman plot suggested underestimation of ΔCBF by NIRS. Divergent associations of pH, pCO2 and arterial oxygen content with rSO2 were found compared to corresponding associations with CBF. In conclusion, NIRS could be a proxy to PET to detect intradialytic CBF changes, although NIRS and PET capture different physiological parameters of the brain

Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion

Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted α-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium

Synthetic Mimic of Antimicrobial Peptide with Nonmembrane-Disrupting Antibacterial Properties

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

Lantibiotic Structures as Guidelines for the Design of Peptides That Can Be Modified by Lantibiotic Enzymes

Lantibiotics are (methyl)lanthionine-containing bacterial peptides. (Methyl)lanthionines are posttranslationally introduced into the prepropeptides by biosynthetic enzymes that dehydrate serines and threonines and couple these dehydrated residues to cysteine residues. Thirty seven lantibiotic primary structures have been proposed to date, but little is known about the substrate specificity of the lantibiotic modifying enzymes. To define rules for the rational design of modified peptides, we compared all known lantibiotic structures by in silico analysis. Although no strict sequence motifs can be defined that govern the modification, statistical analysis demonstrates that dehydratable serines and threonines are more often flanked by hydrophobic than by hydrophilic amino acids. Serine residues escape dehydration more often than threonines. With these rules, novel hexapeptides were designed that either were predicted to become modified or will escape modification. The hexapeptides were fused to the nisin leader and expressed in a Lactococcus lactis strain containing the nisin modifying and export enzymes. The excreted peptides were analyzed by mass spectrometry. All designed fusion peptides were produced, and the presence or absence of modifications was found to be in full agreement with the predictions based on the statistical analysis. These findings demonstrate the feasibility of the rational design of a wide range of novel peptides with dehydrated amino acid residues.