Protein and Peptide Letters - Volume 23, Issue 10, 2016

Ewing’s Sarcoma transcript-2 (EAT2) also known as SH2D1B is involved in regulation of signalling lymphocytic activation molecule (SLAM) family receptor functions. Cytoplasmic tails of SLAM family receptors contain tyrosine residues which mediate the downstream signal transduction through their phosphorylation. EAT2, composed of a single SH2 domain and a short C-terminal tail, binds to the phosphotyrosine residues and regulates SLAM family receptor signalling. We have determined the crystal structure of the human EAT2 protein in an unliganded form. Compared with the mouse EAT2-peptide complex structure, we observe conformational differences in the loops involved in ligand binding. When compared with SAP, the other single SH2 domain protein in human, EAT2 shows similar binding energies to unphosphorylated ligands. This is inconsistent to the previous data showing low affinity of EAT2 toward unphosphorylated peptides compared to SAP which shows high affinity. Additional factors other than the SH2 domains may contribute to the reported differences.

Breast cancer is the most common cancer in women worldwide. Combination of drugs during long-therm cancer therapy can increase free, biological active form of the drug and cause dangerous side effects. The 21st century is a period of searching for a progress in cancer chemotherapy. The simultaneous dosage of drugs and natural agents isolated from fruits and vegetables used in breast cancer treatment could be more effective and less toxic. The aim of the study was to determine the binding sites of both letrozole (LET) and polyphenol product, resveratrol (RES) in tertiary structure of human serum albumin (HSA) based on the fluorescence spectroscopy. The binding of LET and RES to HSA was studied by monitoring the changes in emission fluorescence spectra of albumin in the presence of ligands at 280 nm and 295 nm excitation wavelengths. To identify the binding sites of LET and RES on HSA, warfarin (WAR) and 5-(Dimethylamino)-1-naphthalenesulfonamide (DNSA) were used as site probes for binding site I, while dansyl-L-proline (DP) was studied as a marker for binding site II. The binding sites for LET and RES in HSA were found to be located in subdomain IIIA. Based on the Stern – Volmer and binding isotherm using non-linear regression methods, the formation of complexes of LET and RES with HSA and association constants were obtained. The binding analysis showed that the association constants indicated a stronger interaction of HSA with RES than LET and the presence of RES in the tertiary system alters the stability of LET-albumin complex. This conclusion points to the necessity of precaution and monitoring therapy when resveratrol as a natural compound and letrozole are used together.

The receptor for activated C kinase (RACK) is an important scaffold protein with regulatory functions in cells. However, its role in the immune response of Antheraea pernyi to pathogen challenge remains unclear. To investigate the biological functions of RACK in the wild silkworm A. pernyi, cloning was performed and the expression patterns of the RACK gene were analyzed. Sequence analysis revealed that the RACK gene was 1120 bp containing a 960-bp open reading frame. The deduced RACK protein sequence reveals the higher identity with its homologs from other insects. SDS-PAGE and western blot analysis demonstrated successful expression of a 36-kDa recombinant RACK protein in Escherichia coli. The titer of a rabbit-raised antibody against recombinant RACK protein was about 1: 20000, determined by ELISA. Real-time PCR analysis showed that RACK expression was higher in fat bodies than in other examined A. pernyi tissues. The expression of RACK mRNA in fat bodies of fifth larvae of A. pernyi was obviously induced after nucleopolyhedrovirus, E. coli or Beauveria bassiana challenge. However, the expression patterns of RACK were different in response to these pathogens. Our data suggest that RACK may play a role in the innate immune responses of A. pernyi.

Acetonitrile is a mild solvent, which induces β-sheet conformation in proteins. The global conformational changes in Hb in the presence of ACN were studied using intrinsic fluorescence experiments, acrylamide quenching, ANS fluorescence measurements, soret absorbance spectroscopy, fourier transform infrared spectroscopy, circular dichroism, thioflavin T and congo red assay. Molecular docking showed the binding of hydrophobic residues of Hb to ACN. Hb exists as a partially unfolded intermediate state at 30% v/v ACN. Hb aggregates were obtained at 60% v/v ACN concentration, which were further confirmed by transmission electron microscopy.

The non-enzymatic reaction (glycation) of reducing sugars with proteins has received increased interest in dietary and therapeutic research lately. In the present work, the impact of glycation on structural alterations of camel serum albumin (CSA) by different glucose metabolites was studied. Glycation of CSA was evaluated by specific fluorescence of advanced glycation end-products (AGEs) and determination of available amino groups. Further, conformational changes in CSA during glycation were also studied using 8-analino 1-nephthlene sulfonic acid (ANS) binding assay, circular dichroism (CD) and thermal analysis. Intrinsic fluorescence measurement of CSA showed a 22 nm red shift after methylglyoxal treatment, suggesting glycation induced denaturation of CSA. Rayleigh scattering analysis showed glycation induced turbidity and aggregation in CSA. Furthermore, ANS binding to native and glycated-CSA reflected perturbation in the environment of hydrophobic residues. However, CD spectra did not reveal any significant modifications in the secondary structure of the glycated-CSA. Thioflavin T (ThT) fluorescence of CSA increased after glycation, illustrated cross β-structure and amyloid formation. Transmission electron microscopy (TEM) analysis further reaffirms the formation of aggregate and amyloid. In summary, glucose metabolites induced conformational changes in CSA and produced aggregate and amyloid structures.

Urea and thermal denaturations of bovine serum albumin (BSA) were studied in the absence and the presence of honey or simulated honey sugar cocktail (SHSC) using far-UV CD and ANS fluorescence spectroscopy. Presence of 20% (w/v) honey or SHSC in the incubation mixture shifted the urea transition curve towards higher urea concentrations, being higher in the presence of honey and transformed the two-step, three-state transition into a single-step, two-state transition. A comparison of the far-UV CD and the ANS fluorescence spectra of 4.6 M urea-denatured BSA (U-BSA) in the absence and the presence of 20% (w/v) honey or SHSC suggested greater stabilizing potential of honey than SHSC, as U-BSA maintained native like conformation in the presence of 20% (w/v) honey. Furthermore, thermal transition curves of BSA were also shifted towards higher temperature range in the presence of 20% (w/v) SHSC and honey, showing greater shift in the presence of honey. The far-UV CD spectra of the heat-denatured BSA also showed greater stabilization in the presence of honey. Taken together all these results suggested greater protein stabilizing potential of honey than SHSC against chemical and thermal denaturations of BSA.

ApoAI, a major protein component of HDL is considered to be a key factor which contributes for HDL’s cardio- protective functions. ApoAI is sensitive to myeloperoxidase mediated oxidative modifications under chronic inflammatory conditions such as atherosclerosis. The amino acid tyrosine at position 192 of ApoAI is considered to be one of the vulnerable sites for oxidation which impairs HDL functions and its quality. The presence of oxidized ApoAI in plasma may serve as a useful indicator of CVD risks in humans, but its detection in the clinical settings requires monoclonal antibodies (mAbs) with high specificity. In this study, we have developed mAbs against chloro- tyrosine at position 192 of ApoAI. We designed a small synthetic peptide of 7 amino acids length containing modified tyrosine residue (189-LAE-3-Cl-Y-HAK-195) based on antigenicity prediction. We coupled this peptide to Keyhole Limpet Hemocyanin (KLH) for generating mAbs. Hybridoma clones, KLH-ApoAI-F2 and KLH-ApoAI-H9, were found to be highly specific to chloro-192Tyr containing peptide of ApoAI and not to either nitro-192Tyr containing or to chloro-166Tyr and nitro-166Tyr containing peptides of ApoAI. The utility of these mAbs for screening the quality of HDL as an indicator of CVD risks in humans is discussed.

Neuropeptides and peptide hormones from non-neuronal tissues play important roles in the regulation of insect life. In recent years, the rapid development of analytical techniques has contributed to the discovery of more than 30 families of peptide neurohormones that differ structurally and functionally. Although the discovery of the first neuropeptide occurred almost forty years ago, our knowledge about their full mode of activities, primary structures, synthesis, interactions with receptors or places of action increases gradually and there is still much to unravel. However, one thing is certain. Neuropeptides perform an extremely diverse range of activities. One neuropeptide can affect physiology in different ways. The neuropeptides can act as neurotransmitters, co-transmitters as well as neuromodulators. Most of these molecules have diverse pleiotropic activities on different tissues and organs. Their mode of action includes allatotropic, myotropic, cardiotropic or gonadotropic effects. Activity of some of them is conserved among most of insect species, indicating crucial roles in insect physiology and age of these systems. On the other hand, activity of other neuropeptides and peptide hormones is highly diverse, depending on species or even stages of development. This may indicate that some compounds have taken over the function of others. Insect heart work is regulated in a very complex manner. Myocardium activity undergoes regulation both, by nervous and hormonal way. What is important is that these same compounds can influent on heart as both nervous and hormonal factors. For that reason, the regulation of myocardium is still unclear. In this paper, we summarize the existing knowledge regarding cardioactivity and the involvement of insect neurohormones and some peptide hormones from non-neural tissues to regulation of insect myocardium.

Cancer cells evade apoptosis, which is regulated by proteins of Bcl-2 family in the intrinsic pathways. Numerous experimental three-dimensional (3D) structures of the apoptotic proteins and the proteins bound with small chemical molecules/peptides/proteins have been reported in the literature. In this review article, the 3D structures of the Bcl-2 family proteins from Homo sapiens and as well complex structures of the anti-apoptotic proteins bound with small molecular inhibitors reported in the literature to date have been comprehensively listed out and described in detail. Moreover, the molecular mechanisms by which the Bcl-2 family proteins modulate the apoptotic processes and strategies for designing antagonists to anti-apoptotic proteins have been concisely discussed.

The results of phylogenetic analyses revealed that the family of plant PDI may comprise of at least eight different subfamilies with varying numbers and positions of active centers while retention signals in the endoplasmic reticulum may be present or absent. At least one gene has been cloned for each phylogenetic group. Other phylogenetic analyses have indicated that the family of PDIlike proteins consists of ten classes, the first five of which include proteins equipped with two thioredoxin domains. These results indicate complexity and diversity of the family of protein disulfide isomerase in plants. The study of molecular characteristics of PDI in some cereal species have shown that this enzyme participates in the maturation of secretory proteins and also in the formation of albuminous substances in endosperm, in the mechanism of formation of disulfide bonds and polymerization of gluten polypeptides in wheat. The mechanism of formation of disulfide linkage was tested through in vitro experiments. However, it is not entirely certain whether it reflects their formation in vivo. The results of researches suggest that protein folding and disulfide bond formation occurs in the endoplasmic reticulum. PDI plays a prominent role among enzymes involved in posttranslational modification of proteins. The main goal of this work is to present research data on protein disulfide isomerase, which may be a leading research objective in the area of wheat gluten and the impact of PDI on the baking quality of wheat flour.