Protein and Peptide Letters - Volume 26, Issue 6, 2019

Background: Metabolic processes in living organisms are closely related to the catalytic activity of enzymes. Changes in enzyme activity cause various diseases e.g., neurological, cancer, metabolic and cardiovascular. Most of the current therapeutic drugs available in clinical utilization function as enzyme inhibitors. Objective: The main goal of the current study to contribute to this growing drug design area (such as medication discovery and development) by investigating protein-drug interactions. Methods: The paraoxonase-I (PON1) enzyme was purified from human serum by using different and simple chromatographic techniques. Additionally, it was investigated inhibition effects of some chemotherapeutic drugs on the PON1. Results: The purification results for PON1 depicted a 3880.83 EU/mg proteins specific activity and the molecular weight was calculated as 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These drugs found to strongly inhibit PON1, with IC50 values ranging from 0.222±0.002 to 688.300±0.897 μM. Ki constants for vincristine sulfate, epirubicin hydrochloride, and doxorubicin hydrochloride were determined to be 0.235±0.032 μM, 221.400±29.270 μM, and 913.300±201.000 μM, respectively. Conclusion: These drugs showed in competitive inhibition. Also, the molecular docking poses of these agents inside the catalytic sites of 1V04 and 3SRE were analysis.

Background: Peptide-based therapeutics offer a unique avenue for the development of novel agents for the treatment of diabetes mellitus including α-glucosidase inhibitors. The peptide, SQSPA, was reported to possess to α -glucosidase inhibitory activity in addition to resistance to Gastrointestinal Tract (GIT) digestion. Methods: In this study, the in silico and in vitro structure-activity analyses of the peptide was conducted using alanine scanning to identify key amino acid residues. Results: The alanine scanning led to four analogs viz; AQSPA, SASPA, SQAPA and SQSAA which were GIT stable. Initially, the peptides were subjected to molecular docking on human α- glucosidase and α -amylase where the binding affinities to the enzymes were in the order; AQSPA>SASPA>SQSPA>SQAPA> SQSAA and AQSPA>SQSAA>SASPA>SQSPA> SQAPA, respectively. Hydrogen bond were important for the binding of all peptides but SASPA and AQSPA had the highest hydrogen bonds interactions with the α-glucosidase and α-amylase, respectively. In vitro analysis revealed that the α -glucosidase and α-amylase inhibitory activities of the peptides were in the order AQSPA>SQSPA>SQAPA>SASPA>SQSAA and AQSPA>SASPA> SQAPA>SQSPA>SQSAA, respectively. Using inhibition kinetics, SQSPA was a mixed inhibitor of α-glucosidase while AQSPA, SQAPA and SQSAA showed non-competitive inhibition. For α- amylase inhibition, SQSPA was a non-competitive inhibitor while AQSPA and SQSAA were mixed inhibitors; SASPA and SQAPA showed uncompetitive inhibition. Conclusion: The results indicated that P4 and Q2 are important requirements for the α-glucosidase and α-amylase inhibitory activities of the parent peptide, SQSPA. Furthermore, alanine scanning has led to the design of a novel α-glucosidase inhibitory peptide, AQSPA, with increased activities.

Background: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). Objective: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. Methods: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. Results: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. Conclusion: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.

Background: Antibacterial peptidyl derivative - Cystapep 1, was previously found to be active both against antibiotic-resistant staphylococci and streptococci as well as antibioticsusceptible strains of these species. Therefore, it is a promising lead compound to search for new antimicrobial peptidomimetics. Objectives: We focused on identifying structural elements that are responsible for the biological activity of Cystapep 1 and its five analogues. We tried to find an answer to the question about the mechanism of action of the tested compounds. Therefore, we have investigated in details the possibility of interacting these compounds with biological membrane mimetics. Methods: The subject compounds were synthesized in solution, purified and characterized by HPLC and mass spectrometry. Then, the staphylococci susceptibility tests were performed and their cytotoxicity was established. The results of Cystapep 1 and its analogues interactions with model target were examined using the DSC and ITC techniques. At the end the spatial structures of the tested peptidomimetics using NMR technique were obtained. Results: Antimicrobial and cytotoxicity tests show that Cystapep 1 and its peptidomimetic V are good drug candidates. DSC and ITC studies indicate that disruption of membrane is not the only possible mechanism of action of Cystapep 1-like compounds. For Cystapep 1 itself, a multi-step mechanism of interaction with a negatively charged membrane is observed, which indicates other processes occurring alongside the binding process. The conformational analysis indicated the presence of a hydrophobic cluster, composed of certain side chains, only in the structures of active peptidomimetics. This can facilitate the anchoring of the peptidyl derivatives to the bacterial membrane. Conclusion: An increase in hydrophobicity of the peptidomimetics improved the antimicrobial activity against S. aureus, however there is no simple correlation between the biological activity and the strength of interactions of the peptidyl with bacterial membrane.

Background: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. Objective: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. Methods: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 μM and 20 μM respectively, while 4 μM protein was used for the rest of the experiments. Results: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. Conclusion: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two – state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

Background: The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2. Objective: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein. Methods: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2. Results: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site. Conclusion: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.

Background: A well-known tissue marker of ovarian cancer, Human Epididymis protein 4 (HE4) is the member of whey acidic four-disulfide core proteins family. Purified from human seminal plasma and characterized as a cross-class protease inhibitor, HE4 was proposed to shield spermatozoa against proteolytic factors. However, its exact biological function is unknown. Proteins usually function in conjunction with other proteins in the system and thus, identification and analysis of protein networks become essential to decode protein functions. Objective: This study was performed to explore possible role(s) of HE4 in reproductive physiology via identification of its interactome in human seminal plasma. Methods: HE4 binding proteins were identified through co-immunoprecipitation and MALDITOF/ MS analysis. Also, HE4 was quantified by ELISA in fertile and infertile human seminal plasma samples. Results: Ten HE4 binding proteins were identified, viz. protein phosphatase 1 regulatory subunit 21, protein kinase CLK3, Ankyrin repeat domain-containing protein36A, prostatic acid phosphatase, KIF5C, Spectrin repeat containing, nuclear envelope 1, isoform CRAf, tropomyosin 4, vezatin, utrophin and fibronectin1. This interaction network suggests that HE4 plays multiple roles, specifically in capacitation, sperm motility and maturation. Further, HE4 concentration in human seminal plasma samples was determined by Elisa. Higher HE4 expression in normozoospermia compared to azoospermia and asthenozoospermia affirms its importance in fertilization. Conclusion: Based on identified interactome, it is plausible that HE4 plays a crucial role in fertilization, specifically in sperm maturation, motility and capacitation.