Protein and Peptide Letters - Volume 23, Issue 7, 2016

Mycoplasma pneumoniae is thought to be the simplest and smallest cell wall-deficient bacterium which can cause chronic respiratory infections. Recently vaccination has been a possible and reliable way to reduce the spreading and infection effectively. In this study, the transmembrane proteins P116N (the N-terminal of P116), P1C (the C-terminal of P1), P30, and P116N-P1C-P30 (MP559 for short), a chimeric protein were expressed using prokaryotic expression system. The four purified recombinant proteins were synergized with freund’s adjuvant and immunized New Zealand White rabbits respectively for three times. The IgG antibodies collected from immunized rabbits and mouse were analyzed by ELISA to analyze the immunogenicity and antigenicity. The results showed the four different recombinant proteins could induce strong humoral immune response. Protein MP559 could react with antisera from rabbit immunized with P1C, P30, and P116N, indicating MP559 was well designed and presented antigen epitopes of all the three antigens. Antibodies against P116N, P1C, and P30 could be stimulated by MP559 immunization, indicating MP559 has a potential to replace the three antigens as a vaccine candidate. This study laid a substantial foundation for the vaccine development of M. pneumoniae, and at the same time provided a essential strategy for the vaccine research of other pathogen.

Disulfide C-terminal loop fragments derived from AMPs and the presence of peptidases have been previously reported in the skin secretions of different amphibians. However, there are only a few studies on the identification of enzymes in frog skin secretion based on the primary structure of these proteins. Similarly, little data exist regarding the identification of disulfide C-terminal loops at large scale. Therefore, a comprehensive study on this issue certainly could bring in much more information for understanding this molecular process and its biochemical consequences. Thus, the aim of this work was to characterize the presence of disulfide C-terminal loop fragments of AMPs and identify the proteins and probable enzymes present in the completely unknown secretion contents of the frog Lithobates spectabilis. For this purpose, high-resolution mass spectrometry was applied to analyze the skin secretions processed by two different protocols: (1) using a cocktail of enzymatic inhibitors and 2) without any protease inhibitors, maintaining the solution for 2 hours at 10°C. Results from procedure-1, revealed 122 molecular masses, whereas procedure-2 permitted 253 different molecular masses to be identified. Fifty-nine peptides including 22 disulfide C-terminal loop-containing peptides were obtained following procedure-2. Polyacrylamide gel electrophoresis separation, tryptic digestion and LCMS/ MS were used for “de novo” sequencing of 111 different peptides and the unequivocal identification of fifteen proteins including at least three different peptidases. Additionally, it was possible to fully sequence eight peptides, including a ranatuerin-related peptide identified here as Spectabilin, that was subsequently chemically synthesized and showed high antibacterial, antiparasitic and cytotoxic activities.

Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein–protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer’s disease, Parkinson’s disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

Oxidative damage to the constituents of the eye lens is a major mechanism in the initiation and development of cataract. Lunasin, a 43-amino acids chemoprevention peptide, has been proved to possess potent anti-oxidative activity other than its established anticancer activities. Herein, we explored whether lunasin has preventative effects on d-galactose-induced experimental cataract in rat. After modeling, SD rats were administrated by instillation, 80 μM of lunasin eye drops to each eye thrice daily and consecutively for 30 days. As a result, lunasin treatment effectively inhibited the progression of d-galactose-induced experimental cataract, and protected the lenses of rats from oxidative damage and attenuated the lipid peroxidation through up-regulation of antioxidant enzymes, and inhibited the activation of polyol pathway by decreasing AR activity. Additionally, in vitro studies proved that lunasin treatment could protect human lens epithelial cells (hLECs) against d-galactose induced cell damage and apoptosis, and up-regulate antioxidant enzymes. This is the first demonstration that lunasin could inhibit d-galactose-induced experimental cataract in rats by protecting against oxidative damage and inhibiting the activation of polyol pathway.

The aim of this study was to design and develop stable nanoemulsion formulations containing IFN-γ to probe their use as an immunomodulating agent. The nanoemulsions comprising distilled water, triglycerides of capric acid/caprylic, sobitan-oleate (SP), polysorbate 80 (TW) and propylene glycol (PG) were prepared through ultra-homogenization and characterized regarding droplet size, polydispersity, surface charge, preliminary and accelerated physical stability, and rheological properties. Stable nanoemulsions were selected to incorporate nano doses of IFN-γ (100 ng.mL-1). The influence of IFN-γ incorporated nanoemulsions on functional activity of mononuclear cell for Escherichia coli enteropathogenic was analyzed through superoxide release, phagocytosis, microbicidal activity and intracellular calcium release. The optimized formulation, comprising aqueous and oily phase of 10 % and 80 %, respectively, and surfactant mix ratio (SP/TW/PG) of 3.5/5.5/1.0, remained stable in extreme conditions during 90 days, displaying oil-in-water characteristics, biocompatible pH value, significant maintenance of its rheological profile, hydrodynamic radius of 205 nm, zeta potential of -40 mV and average dose of IFN-γ 91 ng.mL-1. The developed formulation did not alter the MN cell viability rates. Increased the superoxide release, phagocytosis index and intracellular calcium release of MN cells of human blood. Our findings indicate that the produced formulation improved the immunomodulatory activity of the IFN-γ.

The members of plant metallothionein (MT) subfamily p1 are characterized with the presence of six Cys at each end of N- and C-terminal of their amino acid sequences which are arranged in a CXCXXXCXCXXXCXC and CXCXXXCXCXXCXC sequence, respectively. In this study we evaluated the independence of N-terminal Cys-rich region of a type 1 MT isoform from rice (OsMTI- 1b) in forming metal-thiolate cluster. To this end the N-terminal of OsMTI-1b (N-OsMTI-1b) was heterologously expressed in Escherichia coli as fusion protein with glutathione-S-transferase (GST). The E.coli cells expressing GST-N-OsMTI-1b were able to remove Cd2+ and Ni2+ from culture medium. The recombinant GST-N-OsMTI-1b was purified using affinity chromatography. The UV absorption spectra recorded after the reconstitution of the apo-protein with Cd2+ and Ni2+ confirmed that GST-N-OsMTI-1b was able to form complexes with Cd2+ and Ni2+. These results demonstrate the formation of independent metal-thiolate cluster at Nterminal Cys-rich region of GST-N-OsMTI-1b without participation of C-terminal Cys-rich region.

The efficacy of Moringa oleifera seed lectin (WSMoL) as a metal remover in water and the effects of metals on its hemagglutinating and antibacterial activities were determined. Aqueous metal solutions were incubated with WSMoL for 8 h at 4°C and the concentrations of metals were determined using atomic absorption spectroscopy. Hemagglutination and antibacterial assays were conducted with WSMoL and lectin exposed or not to the metals. The removal efficiency of WSMoL was 49.00%, 53.21%, 71.45%, 55.42%, 69.88%, 62.14%, and 49.36% for Cd+2, Pb+2, Cu+2, Zn+2, Mg+2, Mn+2, and Al+3, respectively. WSMoL showed bacteriostatic and bactericidal activities against Escherichia coli and Salmonella enterica serovar Enteritidis. However, hemagglutinating and antibacterial activities were impaired after exposure to metals. In conclusion, WSMoL efficiently removed metals present in water but the interaction with metals impaired lectin carbohydrate-binding ability and antibacterial activity. This should be considered when properties of WSMoL other than metal removal are desired.

Sexual maturation and olfactory bulb defects found in prokineticin 2 (Pk2) and prokineticin receptor 2 (Pkr2) mutant mice resembling the phenotypic characteristics of Kallmann syndrome (KS), gave rise to the question of whether these genes would have a role in KS pathogenesis. Later, mutations in both genes were identified in patients suffering from KS. The gene responsible for the Xlinked form of KS, ANOS1, encodes the ECM protein anosmin 1. Among other functions, anosmin 1 can regulate the activity of FGFR1, encoded by one of the genes involved in the autosomal transmission of KS. Therefore, it has been proposed that anosmin 1 could interact with PKR2 to modulate its activity. We present the first evidence supporting this hypothesis and report the interaction of full-length anosmin 1 with three extracellular domains of PKR2. A truncated anosmin 1 protein comprising the first three domains of the protein interacts with the second extracellular loop of PKR2, involved in PK2 binding. Finally, last three FnIII repeats of anosmin 1 also interacted with the PKR2 domains that interacted with full-length anosmin 1. Our data represent a molecular link between two of the genes involved in KS pathogenesis.

A large variety of vesicles is actively secreted into the extracellular space by most type of cells. The smallest nanoparticles (30-120 nm), called exosomes, are known to transport their cargo (nucleic acids, proteins and lipids) between diverse locations in the body. Specific content of exosomes and their influence on recipient cells depends primarily on the type of the secretory (donor) cell, yet several studies highlight the importance of environmental stress on which the donor cells are exposed. Ionizing radiation, which induces damage to DNA and other structures of a target cell, is one of well-recognized stress conditions influencing behavior of affected cells. A few recent studies have evidenced radiationinduced changes in composition of exosomes released from irradiated cells and their involvement in radiation-related communication between cells. Inducible pathways of exosome secretion activated in irradiated cells are regulated by TSAP6 protein (the transmembrane protein tumor suppressor-activated pathway 6), which is transcriptionally regulated by p53, hence cellular status of this major DNA damage response factor affects composition and secretion rate of exosomes released from target cells. Moreover, exosomes released from irradiated cells have been shown to mediate the radiation-induced bystander effect. Understanding radiation-related mechanisms involved in exosome formation and “makeup” of their cargo would shed light on the role of exosomes in systemic response of cells, tissues and organisms to ionizing radiation which may open new perspectives in translational medicine and anticancer-treatment.

Collagen, a predominant structural protein in extracellular matrix (ECM), is now considered to have probable roles in many biological activities and hence, in different forms have found application as nutraceutical or pharmaceutical therapy option. Many of the biological properties are believed to be due to small hidden peptide residues in the collagen molecules, which come into play after the biodegradation or biosorption of the parent molecule. These peptide regions are called cryptic peptides or by some, as cryptides. The proteolytic hydrolysis of the ECM protein releases the cryptic peptides with many novel biological activities not exhibited directly by the parental protein which include angiogenic, antimicrobial, mitogenic and chemotactic properties. The research for understanding the role of these cryptic peptide regions and making use of them in medical field is very active. Such an understanding could lead to the development of peptide supplements for many biomedical applications. The prolific research in this area is reviewed in this paper.