Modeling of three dimensional structure of human alpha-fetoprotein complexed with diethylstilbestrol: docking and molecular dynamics simulation study

It has been long experimentally demonstrated that human alpha-fetoprotein (HAFP) has an ability to bind immobilized estrogens with the most efficiency for synthetic estrogen analog - diethylstilbestrol (DES). However, the question remains why the human AFP (HAFP), unlike rodent AFP, cannot bind free estrogens. Moreover, despite the fact that AFP was first discovered more than 50 years ago and is presently recognized as a "golden standard" among onco-biomarkers, its three-dimensional (3D) structure has not been experimentally solved yet. In this work using MODELLER program, we generated 3D model of HAFP on the basis of homology with human serum albumin (HSA) and Vitamin D-binding protein (VTDB) with subsequent molecular docking of DES to the model structure and molecular dynamics (MD) simulation study of the complex obtained. The model constructed has U-shaped structure in which a cavity may be distinguished. In this cavity the putative estrogen-binding site is localized. Validation by RMSD calculation and with the use of PROCHECK program showed good quality of the model and stability of extended region of four alpha-helical structures that contains putative hormone-binding residues. Data extracted from MD simulation trajectory allow proposing two types of interactions between amino acid residues of HAFP and DES molecule: (1) hydrogen bonding with involvement of residues S445, R452, and E551; (2) hydrophobic interactions with participation of L138, M448, and M548 residues. A suggestion is made that immobilization of the hormone using a long spacer provides delivery of the estrogen molecule to the binding site and, thereby, facilitates interaction between HAFP and the hormone.     

Correlation between biological activity and conformational dynamics properties of tetra- and pentapeptides derived from fetoplacental proteins

In this work, using molecular dynamics simulation, we study conformational and dynamic properties of biologically active penta- and tetrapeptides derived from fetoplacental proteins such as alpha-fetoprotein, pregnancy specific β1-glycoprotein, and carcinoembryonic antigen. Existence of correlation between flexibility of peptide backbone and biological activity of the investigated peptides was shown. It was also demonstrated that flexibility of peptide backbone depends not only on its length, but also on the presence of reactive functional groups in amino acid side chains that participate in intramolecular interactions. Peptides that demonstrate similar biological effects in regulation of proliferation of lymphocytes and expression of differentiation antigens on their surface (LDSYQCT, PYECE, YECE, and YVCE) are characterized by rigidity of their peptide backbone. Increased backbone flexibility in peptides PYQCE, YQCE, SYKCE, YQCT, YQCS, YVCS, YACS, and YACE is correlated with decreased biological activity. Conformational mobility of amino acid residues does not depend on physicochemical properties only, but also on intramolecular interactions. So, evolutionary restrictions should exist to maintain such interactions in the environment of functionally important sites.     

Dynamic proteomics in modeling of the living cell. Protein-protein interactions

This review is devoted to describing, summarizing, and analyzing of dynamic proteomics data obtained over the last few years and concerning the role of protein-protein interactions in modeling of the living cell. Principles of modern high-throughput experimental methods for investigation of protein-protein interactions are described. Systems biology approaches based on integrative view on cellular processes are used to analyze organization of protein interaction networks. It is proposed that finding of some proteins in different protein complexes can be explained by their multi-modular and polyfunctional properties; the different protein modules can be located in the nodes of protein interaction networks. Mathematical and computational approaches to modeling of the living cell with emphasis on molecular dynamics simulation are provided. The role of the network analysis in fundamental medicine is also briefly reviewed.     

人HCN4通道的滞后现象:影响窦房结起搏的潜在决定因素(英文)

超极化活化环核苷酸门控(hyperpolarization-activated cyclic-nucleotide-gated,HCN)通道参与调制心脏跳动的节律和速率。与HCN1和HCN2有所不同,慢通道HCN4可能不存在电压依赖的滞后现象。本研究采用单细胞膜片钳方法,在稳定转染hHCN4的HEK293细胞上进行电生理记录,观察hHCN4通道是否存在滞后现象,以及cAMP对其的调制作用;同时采用实时定量RT-PCR方法检测窦房结和心房组织中HCNs的表达。电压钳实验结果显示hHCN4电流(Ih)激活随着保持电位超极化的变化而向去极化方向移动。三角电位变化钳(triangular ramp)和动作电位钳的结果也显示了hHCN4的滞后现象。cAMP增加Ih电流幅度,且使电流激活向去极化方向移动,从而改变内源性hHCN4滞后行为。RT-PCR结果显示,人窦房结组织主要表达HCN4,占75%,HCN1占21%,HCN2占3%,HCN3占0.7%。以上结果提示,人窦房结组织主要表达HCN4亚型,hHCN4的Ih存在电压依赖性的滞后现象,且受cAMP调制。由此推断,hHCN4通道的滞后现象可能在窦房结起搏活动中起到了关键作用。     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Conformational dynamics of human α-fetoprotein-derived heptapeptide LDSYQCT analogs

Conformational dynamics of a biologically active fragment of alpha-fetoprotein, the heptapeptide LDSYQCT, and its analogs obtained by site-directed substitutions of amino acid residues were studied. The conformational dynamics of the peptide were conservative under the substitutions Y17F, Y17S, and D15E. Substitutions C19A and S16V resulted only in local changes in the dynamic behavior of the peptide. Chemical modification of cysteine (C19) or dimerization of the peptide by producing a disulfide bond between cysteine residues of two parallel peptide chains, as well as the substitutions C19G, C19S, Q18E, and D15N changed a set of possible conformations and dynamic behavior of all amino acid residues. The most significant changes were caused by substitution of uncharged amino acid residues by charged ones, and vice versa.     

Affinity chromatography of human alpha-fetoprotein on immobilized estrogens

Human alpha-fetoprotein was isolated from abortive material with the help of affinity chromatography on immobilized estrogens. After butanol extraction from the abortive material human AFP obtained the ability for affinity binding with immobilized estrogens. The addition of estrogens to the extract of isolated AFP preparation and incubation with them did not lower AFP binding with immobilized estrogens during the experiments, using affinity chromatography. A 10% buffered aqueous butanol solution was most optimal for elution. The data obtained can suggest that AFP in biological fluids is bound to estrogens, and butanol extraction deestrogenizes human AFP. The mechanism of AFP binding to estrogens in vivo is, evidently, carried out with the help of specific unknown carrier, as AFP does not bind free estrogens.     

Human EGF-derived direct and reverse short linear motifs: conformational dynamics insight into the receptor-binding residues

Short linear motifs (SLiMs) have been recognized to perform diverse functions in a variety of regulatory proteins through the involvement in protein–protein interactions, signal transduction, cell cycle regulation, protein secretion, etc. However, detailed molecular mechanisms underlying their functions including roles of definite amino acid residues remain obscure. In our previous studies, we demonstrated that conformational dynamics of amino acid residues in oligopeptides derived from regulatory proteins such as alpha-fetoprotein (AFP), carcino-embryonic antigen (CEA), and pregnancy specific β1-glycoproteins (PSGs) contributes greatly to their biological activities. In the present work, we revealed the 22-member linear modules composed of direct and reverse AFP_14–20-like heptapeptide motifs linked by CxxGY/FxGx consensus motif within epidermal growth factor (EGF), growth factors of EGF family and numerous regulatory proteins containing EGF-like modules. We showed, first, the existence of similarity in amino acid signatures of both direct and reverse motifs in terms of their physicochemical properties. Second, molecular dynamics (MD) simulation study demonstrated that key receptor-binding residues in human EGF in the aligned positions of the direct and reverse motifs may have similar distribution of conformational probability densities and dynamic behavior despite their distinct physicochemical properties. Third, we found that the length of a polypeptide chain (from 7 to 53 residues) has no effect, while disulfide bridging and backbone direction significantly influence the conformational distribution and dynamics of the residues. Our data may contribute to the atomic level structure–function analysis and protein structure decoding; additionally, they may provide a basis for novel protein/peptide engineering and peptide-mimetic drug design.