Protein and Peptide Letters - Volume 22, Issue 6, 2015

Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the Cterminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.

The effect of reduced (GSH) and oxidized (GSSG) glutathione on the bioactivity of insulin was studied. A polyelectrolyte complex (PEC) of insulin with low molecular weight chitosan (13 kDa) was prepared and characterized. The PEC was then solubilized, in the presence and absence of GSH and GSSG, in a reverse micelle consisting of oleic acid and two surfactants (PEG-8 caprylic/capric glycerides and polyglycerol-6-dioleate). The in vitro and in vivo performances of the reverse micelle formulations (RMFs) were evaluated in rats. At pH 6.5 the association efficiency of the PEC was 76.2%. In vitro insulin release from the RMs was negligible at pH 1.2 and was markedly increased at pH 6.8. The hypoglycemic activity of insulin in the PEC was reduced when administered via the subcutaneous route, regardless of the GSH content. On the other hand, the presence of GSSG significantly enhanced hypoglycemia. When the RMF was administered via the oral route, the presence of GSH had no effect on the hypoglycemic activity of insulin compared with the GSH free system. However, the presence of GSSG in the oral preparation increased the hypoglycemic activity of insulin; probably by inhibiting insulin degradation, thereby prolonging its effect. Thus, incorporation of GSSG in the RMF reduces blood glucose levels in rats and protects insulin from degradation.

Ribosome inactivating proteins (RIPs) are a family of proteins produced by plants, bacteria and fungi. RIPs have specific N-glycosidase activity, and they cleave a specific glycosidic bond in a universally conserved stem and loop structure within the large ribosomal RNA of all organisms. Saporin, a cytotoxic RIP from the plant Saponaria officinalis has been earlier shown to manifest its cytotoxicity by a combination of its N-glycosidase and apoptosis inducing activities. Saporin, along with many other RIPs also has strong inhibitory activity towards HIV integrase. In the current study, using two in vitro model systems, it is established that saporin inhibits propagation of HIV-1 in host cells. Saporin also showed a potent anti-HIV-1 integrase activity in vitro. Using three active site mutants of saporin, which respectively lack N-glycosidase, apoptosis inducing or both activities, it is shown that saporin's in vitro anti-HIV-1 integrase activity is independent of its N-glycosidase activity. However, for the anti-HIV activity of saporin, the apoptosis inducing activity is important.

Behcet's disease (BD) is a recurrent pathema with a typical symptom of inflammation involved in many organs. Previous report indicated that the serum of Korean patients with BD stimulates membrane expression of hnRNP A2/B1 in endothelial cells. In this study, the target 35 kDa recombinant human hnRNP A2/B1 were over-expressed and purified, then sequenced with MALDI-TOF-TOF mass spectrometry. Western blotting and ELISA were applied to detect serum reactivity against hnRNP A2/B1 respectively. The results demonstrate that hnRNP A2/B1 is an autoantigen of BD in Han Chinese population.

Two structurally distinct N-terminal propeptides are known in thermolysin-like proteases (TLPs). Propeptides of the first type are similar to the prosequence of thermolysin, while the second type propeptides resemble the protealysin propeptide. At the same time, the catalytic domains of all enzymes of the family are highly similar. The available data suggest that the propeptides determine the biological function of TLPs. It was shown that the thermolysin-type propeptides act as folding assistants, can inhibit cognate mature proteins, and influence their secretion. However, the functions of protealysin-like propeptides remain unclear. Here, we studied the effect of the propeptide on protealysin folding for the first time. After heterologous expression in E. coli cells, active enzyme is formed only in the presence of the propeptide either in cis or in trans. Thus, both types of TLP prosequences can function as folding assistants despite their structural dissimilarity and absolutely different interaction with the cognate catalytic domains as indicated by X-ray data.

Determining the substrate specificity of a protease is essential for developing assays, inhibitors and understanding the mechanisms of the enzyme. In this work, we have profiled the specificity of Peptidyl-Lys metallopeptidase, (LysN), of Armillaria mellea, by a synthetic fluorescence resonance energy transfer (FRET) positional-scanning library. The library was based on a reference sequence K(Abz)-S-A-Q-K-M-V-S-K(Dnp), where the fluorescent donor is 2-aminobenzamide and the quencher is N-2,4-dinitrophenyl. Each position was varied between 19 different amino acids one by one, to reveal the specificity of the protease. LysN exhibits strict specificity for lysine in S1’, and has less specificity moving further away from the scissile bond. Additivity between the subsites was observed and the best substrate identified was K(Abz)-M-R-F-K-R-R-R-K(Dnp) with a kcat/KM of 42.6 µM/s. Based on a homology structure model the reference substrate was fitted into the active site using molecular dynamics to propose peptide-enzyme interactions.

Cathepsin E is an aspartic protease that belongs to the pepsin family. This protease is similar to cathepsin D but differs in its tissue distribution and cell localization. Elevated levels of this enzyme are linked to several tumors, including devastating pancreatic ductal adenocarcinoma. In this manuscript, we present a new protocol for the high-yield purification of recombinant human cathepsin E in the baculovirus expression system. The recombinant protein was produced by the Sf9 insect cell line and secreted into the medium in the form of an inactive zymogen. Procathepsin E was purified using ion-exchange and size exclusion chromatographies followed by pepstatin- and heparin-affinity chromatography steps. The zymogen was activated at an acidic pH, resulting in a high yield of the activated intermediate of cathepsin E. The enzymatic activity, stability, and molecular weight corresponded to those of cathepsin E. The new purification procedure will promote further studies of this enzyme to improve the understanding of its structure-function relationship and consequently enable the development of better therapeutic approaches.

After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins.

PP2A is a serine/threonine phosphatase critical to a number of physiological and developmental processes. In this manuscript, we show that a peptide, specifically blocking the caspase- 9/PP2A interaction, DPT-C9h, induces apoptosis in primary tumour B cells isolated from peripheral blood mononuclear cells or bone marrow of chronic lymphocytic leukemia (CLL) patients, but not on B cells obtained from healthy donors (HD). Moreover, in both CLL patients and HD, DPT-C9h does not induce apoptosis on T- and NKcells and monocytes. Our results strongly suggest that DPT-C9h peptide has tumour specificity and that caspase-9/PP2Ac interaction constitutes a novel therapeutic approach for the treatment in CLL patients.

Most published articles always applied a certain model or arithmetic to only a certain dataset. Considering the avalanche of biological data created in the post-genomic age, this type of research shows many shortcomings and inefficient characteristics, because it is always have difficulties to apply the same model to different datasets. So we proposed a multifunctional ensemble classifier which combines several individual classifiers. Each of them was trained in different parameter system. The final outcomes were combined through a weighted voting system. This classifier was conducted on several strictly constructed biological datasets. Based on the testing result from three different types of biological dataset, this new predictor can deal with more sweeping range of biological data, and receives more efficient and robust results in comparison with other published methods tentatively.

An endo-pectate lyase (PL1B) of family 1 polysaccharide lyase from Clostridium thermocellum was structurally characterized and its stability under chaotropic agent was determined. The putative domain PL1B was identified from the protein sequence ABN53381.1 belonging to superfamily 3 of pectate lyase. Multiple sequence alignment of PL1B with other known pectate lyases revealed the conserved and semi-conserved residues. The secondary structure of PL1B predicted by PsiPred and confirmed by Circular Dichroism showed the presence of 2 α-helices (2.06%), 26 β-strands (40.54%) and 29 random coils (57.4%). The modelled protein represented right handed parallel β-helix structure, where three parallel β-sheets linked by loops coils around to form the β-helix core. Quality assessment of energy minimized structure by Ramachandran plot displayed 82.8% residues in favoured region. Superposition of PL1B structure with Bsp165-PelA from Bacillus sp. revealed the substrate binding cleft formed by the amino acid residues from the loops and β-sheet. Molecular dynamic simulation of modelled PL1B structure inferred that it is quite stable and compact. Docking studies identified Asp151, Arg209, Asn234, Arg236, Tyr271 and Ser272 as the key residues of PL1B involved during catalysis. Among them Arg209 is responsible for proton abstraction during β-elimination. Protein melting studies on PL1B showed that there was 12°C shift of peak from 74 to 86°C in presence of 0.6 mM Ca2+ ions, showing that they provide stability to the structure. The unfolding of PL1B by GuHCl or Urea by fluorescence study showed that the protein structure is stable and disintegrates at their higher concentrations.