Protein and Peptide Letters - Volume 24, Issue 10, 2017

Antimicrobial peptides (AMP) inhibit the proliferation of bacteria and frequently protect experimental animals from bacterial challenge. If the mode of action is membrane disintegration, one would expect that AMP can also kill cancer cells whose membrane structure lies between those of normal and bacterial cells. However, an ever-increasing number of reports suggest that AMP, with their newer name, host-defense peptides (HDP), do not directly kill bacteria under in vitro conditions when small molecule antibacterials are bactericidal. The micromolar activity may be suitable for biochemical studies but does not warrant oncology drug development. Nevertheless, as HDP are also documented to act on intracellular targets, the alternative modes of action revive the belief that antiproliferative efficacy can be obtained, indeed supported by a few successful animal efficacy studies. In addition, the passive transport properties of AMP/HDP can be utilized in the intracellular delivery of unrelated cancer drugs. Unfortunately the inherent pro-inflammatory activities of many native and designer HDP lead to oncogenic rather than anti-cancer activities in vitro and in vivo. A critical evaluation of the role of HDP in tumor development with pharmaceutically relevant animal efficacy and toxicity studies are needed before human clinical trials can be designed and initiated.

Background: Type 2 diabetes mellitus (T2DM) is the most common lifestyle disease affecting all countries. Due to its asymptomatic onset, it is often diagnosed after irreversible vascular complications have been initiated. Therefore, specific markers characteristic for very early disease stages and suitable for early diagnostics are required. Glycation of plasma proteins, such as human serum albumin (HSA), has been often suggested as marker. However, the total glycation degree of HSA does not provide sufficient information about short-term fluctuations of blood glucose concentrations due to the large number of glycation sites. Analysis of individual modification sites might be more informative, but methods for reliable quantifications are still missing. Objective: The main objective of this study was to establish and qualify a method of analysis applicable to sensitive and precise quantification of glycations sites in plasma proteins. Methods: Plasma samples obtained from diabetic patients and non-diseased individuals were separated from low-molecular weight compounds, digested with trypsin, enriched for glycated peptides by boronic acid affinity chromatography (BAC), desalted by solid phase extraction (SPE), and separated by RP-HPLC coupled online to ESI-QqQ-MS. Quantification relied on multiple reaction monitoring (MRM) of multiple glycation sites identified in plasma proteins using a stable isotope dilution approach or internal standardization. Results: The data presented here suggests high selectivity and precision (relative standard deviations below 10%) of the overall approach appearing to be well suited for the identification of prospective biomarkers. Six glycated peptides corresponding to different glycation sites of HSA were present in plasma samples obtained from T2DM patients at significantly higher levels than in non-diabetic men matched for age. Additionally, each of the studied glycation site of HSA appeared to be affected at different degrees. Conclusion: The presented approach enables the sensitive and robust quantification of prospective T2D biomarkers promising for clinical diagnostics.

Background: Soybean is a nutritionally important source of proteins and oils. Organspecific analyses of the plants provided valuable information about physiological changes occurring in particular organ. However, studies on the vegetative stage soybean during the growth and development are lacking. Analyzing the growth stages of soybean at proteomic level is particularly important for understanding the growth dynamics. Objectives: The main objective of this study was to investigate the organ-specific growth pattern of proteins during early growth stage. Methods: Soybean (Glycine max L. cv Enrei) seeds were sown in each seedling case for 10 days and roots, hypocotyls, and leaves were collected. Proteins were extracted and analyzed by nano-liquid chromatography mass spectrometry. Mole percent abundance was calculated using emPAI values. To determine the functional role of the proteins identified in the MS analysis, functional categorization was performed using MapMan bin codes. Visualization of protein abundance ratio was performed using MapMan software. Enzyme activity and quantitative reverse transcription-polymerase chain reaction analyses were performed. Results: A total of 357, 360, and 392 proteins were identified in root, hypocotyl, and leaf of vegetative stage soybean, respectively. Proteins related to stress, cell organization, transport, signaling, and mitochondrial electron transport chain decreased in root, hypocotyl, and leaf. Proteins related to protein metabolism, glycolysis, and cell wall were comparable in root and hypocotyl; however, in leaf, glycolysis and cell wall related proteins were decreased. Aldehyde dehydrogenase was decreased in abundance and activity in hypocotyl and leaf as compared to root. Major latex proteins 43 and 423 changed in abundance in an organ-specific manner. The mRNA expression level of major latex proteins exhibited a differential expression in the hypocotyl of soybean during flooding stress and recovery. Conclusion: Our results suggest that aldehyde dehydrogenase and major latex proteins play key roles in growth of soybean in an organ-specific way.

Background: Chronic liver disease is often associated with the infection by hepatitis C virus (HCV), which is an enveloped RNA virus belonging to the Flaviviridae family. Many studies found that milk proteins, such as lactoferrin, might have profound antiviral activity against HCV. Various secretory fluids ranging from milk, to tears, saliva, and nasal secretion, and to bile and pancreatic juice, as well as neutrophils, mucosal surfaces, and blood contain a widely spread multifunctional glycoprotein, lactoferrin (Lf), structure of which can be depicted as two homologous domains connected by the short spacer peptide. Objective: This study aimed to understand the effectiveness of the synthetic peptides cLfsp, bLfsp, hLfsp1, and hLfsp2 corresponding to the spacer peptides of camel, bovine, and human Lfs, respectively, against HCV in in vitro settings. Method: We used RT-nested PCR to evaluate the antiviral activity of the synthesized spacer peptides against HCV infectivity in PBMC and HepG2 cells looking at their neutralization, protection, and intracellular treatment potentials. Results and Conclusion: We show that direct interaction of hLfsp1, hLfsp2, and bLfsp with viral particles is able to neutralize the HCV entry into HepG2 cells (with hLfsp2 being more potent neutralizer than hLfsp1 and bLfsp), whereas cLfsp does not show any neutralizing potential. Therefore, our analysis revealed that different spacer peptides are characterized by different antiviral potentials and use different mechanisms for antiviral protection.

Background: Glutathione transferases (GSTs, EC. 2.5.1.18) form a large group of multifunctional enzymes that are involved in the metabolism and inactivation of a wide range of endogenous and xenobiotic compound as well as in cell regulation and response to several biotic and abiotic stresses. Objectives: In the present work, we report the comparative analysis of the structural and functional features of two isoenzymes (GmGSTU5-5 and GmGSTU8-8) of the glutathione transferase (GST) family from Glycine max. Methods: Full-length cDNA clones of GmGSTU5-5 and GmGSTU8-8 were derived from RT-PCR of RNA isolated from soybean seedlings and were cloned into a T7 expression vector. he recombinant enzymes were expressed in E. coli and purified by affinity chromatography. Substrate specificity, kinetic and inhibition analysis were carried out towards a range of different xenobiotic compounds and GSH analogues. The thermal stability of the enzymes was also evaluated using activity assays and differential scanning fluorimetry. Results: Analysis of substrate specificity using a range of thiol substrates and electrophilic compounds suggested that both isoenzymes display broad and overlapping specificities. They are capable of detoxifying major stress-induced toxic products. Study of their ligandin-binding properties by kinetic analysis and molecular modelling indicated that both GmGSTU5-5 and GmGSTU8-8 bind a range of secondary metabolites and plant hormones, suggesting a role in transport or storage of bioactive compounds. Thermostability analysis showed that GmGSTU5-5 and GmGSTU8-8 display extraordinary thermal stability, compared to other plant GSTs. Conclusion: Our results suggest that GmGSTU5-5 and GmGSTU8-8 display different or overlapping substrate specificities and kinetic properties. The biological role of GmGSTU5-5 and GmGSTU8-8 may be relevant to the detoxification of toxic compounds or the binding of bioactive metabolites that function in cell regulation and stress defence mechanisms.

Background: Urokinase type plasminogen activator (uPA) is a 53-kDa serine protease initially synthesized as a catalytically inactive single chain polypeptide. Inactive-uPA is subject to proteolytic cleavage, which results in the two-chain active protein. uPA plays key roles in the enhancement of cell malignancy during tumor progression. Objectives: The main objective of this review was to analyze and describe the main molecular mechanisms involved in the regulation of uPA expression in cancer Methods: Searching literature to evaluate and define the relevant information regarding to the state of the arts on uPA functionality and regulation in cancer, including intracellular signaling regulation, tumor progression, invasion, epigenetic mechanism, and finally uPA as therapeutic target in cancer. Results: uPA expression is dysregulated in tumor cells, which results in increased cellular invasion capacities reflecting changes in uPA activity and expression during tumor progression. In this review we discuss the main aspects of uPA, from its capacity to activate plasminogen to plasmin, to the main intracellular signal transduction mechanisms as well as the epigenetic mechanisms involved in the regulation of uPA expression, including regulation by microRNAs. As well as, the current therapeutic methodologies targeting uPA for cancer treatment are described. Conclusion: Although, uPA is dysregulate in tumor progression, its expression is finely regulated at both enzymatic activity and at protein expression as well, which allow cancer cells efficiently survive, proliferate, and spread into neighbouring tissues and distant organs. Moreover, since uPA implications in tumor development and cancer cell invasion and metastasis, it is an attractive target for cancer chemotherapies.

Background: Melanoma-associated antigen-A3 (MAGE-A3) is a tumor specific antigen and a potential candidate for cancer immunotherapy. We had screened three immunodominant multiepitopes of MAGE-A3, and identified these multiepitope peptides had significantly higher reactivity to serum samples from gastric cancer patients. However, the immune responses of three multiepitope peptides carried by HBcAg in mice have not been investigated. Objectives: The main objective of this study was to analyze the humoral and cellular immune responses in mice induced by these three multiepitope vaccines of MAGE-A3. Methods: Three multiepitopes of MAGE-A3 (MAGE-A3(EPI-1, or -2, or -3)) were respectively inserted at HBcAg major immunodominant region (HBcAg(MIR)) of the pET21a(+)/HBcAg(MIR) recombinant plasmid. These recombinant chimeras were identified by PCR, and transfected respectively into E. Coli Ressotta strain. The expression products of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) were purified respectively by Ni2+ chelated affinity column, and then confirmed by SDS-PAGE and Western-blot analysis.Purified three rHBcAg(MIR)/MAGE-A3 multiepitopes were administrated respectively into BALB/c (H-2Kd) mice by intradermal injection. The production of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) specific IgG in serum from immunized mice were measured by ELISA. Spleen cells from all immunized mice were harvested after one week of last immunization for lymphocyte proliferation assay and cytotoxic T-lymphocyte assay. Results: PCR and Sequencing analysis showed the presence of the required gene fragment in pET21a(+)/ HBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) recombinant plasmid. Purified rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) could be probed specifically by McAb of 6is-tag. ELISA analysis indicated that serum from immunized mice with rHBcAg(MIR)/MAGE-A3(EPI-1, -2, or -3) proteins could be discerned specifically by complete MAGE-A3 protein, and high level of antibodies in immune serum were obtained, and all antibody titers could reach above 1:1600. The splenocytes from groups of rHBcAg(MIR)/MAGE-A3(EPI-1,-2, or -3), stimulated respectively with corresponding peptides showed the higher proliferative responses comparing with control groups of HBcAg(MIR) or PBS (p<0.05, respectively). Splenocytes from mice immunized with rHBcAg(MIR)/MAGE-A3 (EPI-1, or -2, or -3) could killed target cells effectively, and there were significant difference of CTL activities compared with control groups of HBcAg(MIR), or PBS (p<0.05, respectively) at any ratio of effector : target. Conclusion: Our results indicated MIR in HBcAg presenting platform could present MAGE-A3 multiepitopes efficiently and induced significant humoral or cellular immunity. The immune strategy based on multiepitopeimmunization could have potential for preventing or controlling MAGE-A3 associated malignant disease.

Background: Proteins tend to form inactive aggregates under harsh conditions used in industrial processes. Lipases are enzymes that hydrolyse triglycerides to glycerol and free fatty acids, but are able to catalyse various other transformations in the presence of organic solvents. Objectives: The main objective of this study was to investigate lipases behavior at high temperature and in presence of organic solvents. Methods: Heat-induced aggregation of porcine pancreatic lipase (PPL) was followed by UV-visible spectroscopy at 400 nm wavelength for 600 seconds, at the isoelectric point (pH 5, phosphate solution) and 50°C, and in presence or absence of various percentages of dimethyl sulfoxide (DMSO), propanol, isopropanol, acetone and trifluoroethanol (TFE). Possible positioning of each organic solvent molecule relative to PPL was investigated using docking method. Results: Native enzyme aggregated under aforementioned conditions and amorphous aggregates formed which were visible to the naked eye. From the tested solvents, DMSO reduced protein aggregation in a concentration-dependent manner. On the other hand, protein aggregation intensified by adding any of propanol, isopropanol, acetone or TFE. This effect was more pronounced in TFE and propanol compared to isopropanol and acetone. Conclusion: Solvents with lower polarity led to aggregation, while solvent with higher polarity inhibited PPL aggregation, and DMSO could be effectively used to counteract lipase aggregation.

Background: The Escherichia coli chaperonin GroEL represents the paradigmatic molecular machine of protein folding. Most of our knowledge on GroEL function is derived from studies with denatured water-soluble proteins or short peptide mimetics. In our earlier studies, we observed that newly translated membrane protein CXCR4 gained significant folding enhancement upon interacting with GroEL in the presence of ATP and GroES. This highlights the mechanistic flexibility and substrate diversity of the chaperonin. Objectives: This work extends our previous observation to the study of binding of GroEL with CXCR4 transmembrane peptides. Methods: The model peptide corresponding to the first transmembrane α-helix in the native tertiary structure of CXCR4 was commercially synthesized. A fluorescent tag was attached to the Nterminus of the peptide for ease of fluorescence characterization. Binding of CXCR4 peptide to GroEL was investigated by fluorescence anisotropy as well as isothermal titration calorimetry (ITC). Through model fitting to the anisotropy and ITC data, important thermodynamic parameters were obtained for the binding. In identifying the binding site of GroEL apical domains for the CXCR4 peptide, competitive binding of the peptide and a model “strongly binding peptide” (SBP) was performed by ITC. Furthermore, the kinetics of the CXCR4 peptide binding to GroEL was also studied by anisotropy. Results: Through anisotropic measurement of binding of CXCR4 peptide to GroEL, a dissociation constant and binding stoichiometry of 0.10μM and 7.00 was obtained. Thermodynamic parameters of the binding were also determined by ITC. By fitting the ITC data to one set of sites model, the values of the thermodynamic parameters were acquired directly as follows: ΔH=-627.10kcal/mol; ΔS=-186.23cal/mol; KD=0.23μM; N=5.96. The binding site of the CXCR4 peptide in GroEL was also probed through competitive binding with the model peptide SBP, pointing to the groove between paired α helices H and I in the apical domain. In addition, the binding kinetics suggests a slow dissociation of the peptide-GroEL complex. Conclusion: The CXCR4 model peptide has been shown to bind to GroEL with high affinity. The binding stoichiometry was estimated to be 6 or 7 depending on the analysis method. Furthermore, the two thermodynamic parameters ΔH and ΔS that define the binding affinity have also been measured by ITC. The binding site of the CXCR4 peptide in the GroEL apical domain was investigated through competitive binding studies with the model peptide SBP. Meanwhile, the kinetic studies indicate a slow dissociation of the peptide-GroEL complex. These results obtained by the reduced approach of employing CXCR4 transmembrane peptides would be beneficial to understanding the GroEL-CXCR4 interaction in the assisted folding.

Background: Misfolding of proteins often leads to aggregation. Accumulation of diverse protein aggregates in various cells, tissue and organs is the hallmark of many diseases, such as Alzheimer's disease and Parkinson's disease. Objectives: The main objective of this study was to present a novel method of characterization of protein aggregates, associated with differential toxicity with different size and composition in vitro using flow cytometry. Methods: A Beckman Coulter Epics XL flow cytometer with argon ion laser operating at 488 nm was used for flow cytometry analysis. The voltage and the gain settings for individual channels were set at high voltage and gain for the detections of autofluorescence, fluorescence of adsorbed Congo red, forward scattering (FSC) and side scattering (SSC) intensities from the aggregates of proteins and nanoparticles. Each sample was analyzed to characterize and quantify the number of aggregates with a limit of maximum 20,000 events. The flow cytometry data were analyzed using Flowing software version 2.5.1 and Origin 8.0. Results: Autofluorescence and scattering intensities could distinguish between amyloid and nonamyloid aggregates. Dot plots of both side scattering (SSC) and forward scattering (FSC) intensities also showed characteristic fingerprint of both the types of aggregates when compared with those of well known nanoparticles of oxides of Fe and Cu. Conclusion: This work reports a novel, simple and robust flow cytometric method of characterization of protein aggregates of different size and composition which would find wider application in characterization of biomolecular aggregates, in general.

Background: Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. Objectives: The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. Methods: The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. Results: The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. Conclusion: The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

Background: The serological diagnostic methods currently available for mucocutaneous leishmaniasis (MCL) lack specificity when complete parasites are used; however, such specificity increases when protein fractions are used. Ribosomal proteins have been reported to induce antibodies in animal and humans infected with the parasite, making them a worth candidate to assess its diagnosis potential. Objective: This study was thus aimed at evaluating synthetic peptides derived from Leishmania braziliensis ribosomal proteins S25 and S5 as antigen candidates for diagnosing MCL by ELISA Methods: It was used 8 and 13 peptides derived from ribosomal proteins 25 and S5 respectively as antigens in order to detect IgG antibodies by ELISA in people with active MCL, Chagas disease (CH) and autoimmune disease (AID). Results: 4 of these 21 peptides (P4, P6, P19 and P21) had the greatest sensitivity (21.7%, 13.04%, 20% and 20%, respectively) as well as having 95%, 100%, 100% and 82.5% specificity, respectively. Conclusion: The study revealed the limited usefulness of the peptides being studied as a diagnostic tool in the conditions used here, because its low sensitivity, but it is worth highlighting that the use of peptides as antigen in the serodiagnosis of MCL may overcome the cross reaction presented with other antigens, thus avoiding false positives.