Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Autocatalytic nature of "slow virus infections"

A concept that considers the causative nature of the so-called "slow virus infections", causing syndromes of spongiform encephalopathies in man and animals as a chain autocatalytic process is put forward. According to this concept, PrP(27-30) protein, isolated recently from the brains of scrapie-infected animals, is a C-terminal domain of the normal protein component of brain tissue which is a latent zimogen. Certain clinical and experimental data are discussed within the framework of this concept. Exogenous proteinases are presumed to be capable of triggering such a chain autocatalytic process in the brains of susceptible animals. Indeed, in one of our experiments, a subtoxic dose of pronase injected into mouse brain induced the development of a syndrome indistinguishable from spongiform encephalopathy in its clinical and pathomorphological manifestations. The probable role of neuron-specific proteins of intermediate filaments in such pathological processes is discussed. It seems possible that spongiform encephalopathies are particular cases of pathological processes that have catalytic nature. Presumably, the Alzheimer disease has such a catalytic causative nature.     

Preparation of pro-oncogenic mutant forms V659E and V659Q of the transmembrane domain of receptor protein kinase ErbB2 for structural studies

Receptor tyrosine kinases (RTKs) play an important role in intercellular signal transduction through the plasma membrane. RTKs are integral membrane proteins activated upon lateral homo- or heterodimerization involving their transmembrane domain. The polymorphism and mutations in RTK transmembrane (TM) domains are directly associated with a number of human diseases. The family of epidermal growth factor receptors, ErbB, is an important class of RTKs participating in human cell growth, development, and differentiation. In order to investigate the influence of pathogenic mutations in ErbB TM domains on the structural and dynamic properties of these receptors and on specific interactions of their TM domains, we have developed highly effective systems of bacterial expression and purification of recombinant transmembrane fragments ErbB2_641–684 with pro-oncogenic substitution of Val⁶⁵⁹ by Glu or Gln. Transmembrane fragments were obtained in Escherichia coli BL21 (DE3) pLysS as a fusion protein with thioredoxin A. The purification protocol includes immobilized metal ion affinity chromatography (IMAC) and cation-exchange chromatography. The application of the protease Thrombin for hybrid protein hydrolysis considerably reduces financial expenditure as compared to the analogous protocols. The described techniques allow obtaining the milligram quantities of ErbB2 transmembrane fragments and its ¹⁵N-/[¹⁵N, ¹³C]-isotope-labeled derivatives for the analysis of their spatial structure using high-resolution heteronuclear NMR spectroscopy in a membrane-mimicking milieu.     

Tunable Goos-Hänchen Shift of Surface Plasmon Beam Due to Graphene in a Metal-Dielectric System

The tunneling of surface plasmon waves between two slabs of dielectric prisms superposed on the metal surface is studied. The prism with the incident surface plasmon wave is superposed by a stack of graphene sheets. The analytical theory is built to connect the Fermi energy of graphene with the Goos-Hänchen shift of the transmitted surface plasmon waves. The obtained results may be useful for developing integral switching devices on the basis of surface plasmon polaritons.

     

Antiamoebin I in methanol solution: rapid exchange between right-handed and left-handed 3(10)-helical conformations

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe(1)-Aib-Aib-Aib-Iva-Gly-Leu-Aib(8)-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl(16). By using the uniformly (13)C,(15)N-labeled sample of Aam-I, the set of conformationally dependent J couplings and (3h)J(NC) couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the (13)C(beta) nuclei (Deltadelta((13)C(beta))) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe(1)-Gly(6)) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3(10)-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib(8) residue. The (15)N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib(8) residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic 'propensity' possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.     

In vivo and in vitro estimations of the direct effect of estrogen on rat hepatocytes tested by the changes in the unusual estrogen-binding protein content

The direct effect of estradiol (E2) on the hepatocytes of mature male rats has been examined by measuring the changes in the unusual estrogen-binding protein (UEBP) content and parallel measuring the level of liver estrogen receptors (ER). The content of UEBP (NUEBP) and ER (NER) in the liver were determined using the quantitative methods for differential specific determination of the E2-binding sites of these proteins. It has been shown that the administration of E2 in vivo induced a considerable decrease in hepatic NUEBP not only in intact males, but also in hypophysectomized males during the initial period after the operation (when the content of hepatic ER was still high) and produced no effect in hypophysectomized males during the later period (when liver ER were depleted). Repeated administration of human growth hormone (hGH) (twice a day) resulted in a considerable increase in NER in hypophysectomized males and restored the sensitivity to the subsequent inhibitory effect of E2 on UEBP. We also used rat hepatocytes after a 4-day primary culturing. These cells had a stable morpho-functional status, high ER level, and sex-differentiated UEBP content. Culturing of mature male rat hepatocytes in the medium containing E2 at concentrations close to physiological levels (10(-10)-10(-7) M) decreased NUEBP in a dose-dependent manner. Hexestrol (10(-7) M) but not cholesterol (10(-5) M) also exhibited a direct effect on NUEBP in cultured rat hepatocytes. The effect of E2 was reversible: statistically significant increase in NUEBP was observed 3 days after 10(-9) M E2 had been removed from the culturing medium. It was concluded that hepatocytes may be a primary target for E2 under physiological conditions and that GH may modulate the direct effect of E2 at the hepatic level by modifying the content of liver ER.     

Characterization of a 95 kDa high affinity human high density lipoprotein-binding protein

A new human 95 kDa high density lipoprotein (HDL)-binding protein (HBP) corresponding to a high affinity HDL-binding site with K(d) = 1.67 microg/mL and a capacity of 13.4 ng/mg was identified in human fetal hepatocytes. The HDL binding with the 95 kDa HBP plateaus at 2.5-5 microg/mL under reducing and nonreducing conditions. The association of HDL(3) with the 95 kDa HBP plateaued in 15-30 min while dissociation was complete in 30 min. HDL(3), apoA-I, and apoA-II were recognized by the 95 kDa HBP while low density lipoproteins (LDL) and tetranitromethane-modified HDL were not. The 95 kDa HBP predominantly resides on the surface of cells since trypsin treatment of HepG2 cells eliminated nearly 70% of HDL binding. All studied human cells and cell lines (HepG2, Caco-2, HeLa, fibroblasts, SKOV-3, PA-I) demonstrated the presence of the 95 kDa protein. Both RT-PCR and Western blotting for HB-2/ALCAM were negative in human fetal hepatocytes while Gp96/GRP94 was clearly differentiated from the 95 kDa HBP by two-dimensional electrophoretic mobility. Moreover, deglycosylation of HepG2 membrane preparations did not affect either HDL binding to the 95 kDa HBP or its size, while in contrast it affected the molecular weights of HB-2/ALCAM and SR-BI/CLA-1. We conclude that the 95 kDa HBP is a new HDL receptor candidate widely expressed in human cells and cell lines.     

Membrane binding motif of the P-type cardiotoxin

Carditoxins (CTXs) from cobra snake venoms, the basic 60-62 residue all-beta sheet polypeptides, are known to bind to and impair the function of cell membranes. To assess the membrane induced conformation and orientation of CTXs, the interaction of the P-type cardiotoxin II from Naja oxiana snake venom (CTII) with perdeuterated dodecylphosphocholine (DPC) was studied using ( 1 )H-NMR spectroscopy and diffusion measurements. Under conditions where the toxin formed a well-defined complex with DPC, the spatial structure of CTII with respect to the presence of tightly bound water molecules in loop II, was calculated using the torsion angle dynamics program DYANA. The structure was found to be similar, except for subtle changes in the tips of all three loops, to the previously described "major" form of CTII in aqueous solution illustrated by the "trans" configuration of the Val7-Pro8 peptide bond. No "minor" form with the "cis" configuration of the above bond was found in the micelle-bound state. The broadening of the CTII backbone proton signals by 5, 16-doxylstearate relaxation probes, together with modeling based on the spatial structure of CTII, indicated a periphery mode of binding of the toxin molecule to the micelle and revealed its micelle interacting domain. The latter includes a hydrophobic region of CTII within the extremities of loops I and III (residues 5-11, 46-50), the basement of loop II (residues 24-29,31-37) and the belt of polar residues encircling these loops (lysines 4,5,12,23,50, serines 11,46, histidine 31, arginine 36). It is suggested that this structural motif and the mode of binding can be realized during interaction of CTXs with lipid and biological membranes.     

Low level viral persistence after infection with LCMV: a quantitative insight through numerical bifurcation analysis

Many important viruses persist at very low levels in the body in the face of host immunity, and may influence the maintenance of this state of 'infection immunity'. To analyse low level viral persistence in quantitative terms, we use a mathematical model of antiviral cytotoxic T lymphocyte (CTL) response to lymphocytic choriomeningitis virus (LCMV).This model, described by a non-linear system of delay differential equations (DDEs), is studied using numerical bifurcation analysis techniques for DDEs. Domains where low level LCMV coexistence with CTL memory is possible, either as an equilibrium state or an oscillatory pattern, are identified in spaces of the model parameters characterising the interaction between virus and CTL populations. Our analysis suggests that the coexistence of replication competent virus below the conventional detection limit (of about 100 pfu per spleen) in the immune host as an equilibrium state requires the per day relative growth rate of the virus population to decrease at least 5-fold compared to the acute phase of infection. Oscillatory patterns in the dynamics of persisting LCMV and CTL memory, with virus population varying between 1 and 100 pfu per spleen, are possible within quite narrow intervals of the rates of virus growth and precursor CTL population death. Whereas the virus replication rate appears to determine the stability of the low level virus persistence, it does not affect the steady-state level of the viral population, except for very low values.     

New recombinant variant of human immunodeficiency virus of type 1, subtype envB/envA, isolated in Novosibirsk

The nucleotide sequence of the variant of human immunodeficiency virus of type 1 (HIV-1), mostly widespread on the territory of the Novosibirsk region, was determined. The analysis of the nucleotide sequence confirmed that this variant belonged to HIV-1 of subtype A. The HIV-1 recombinant variant of subtype envB/envA with the recombination area within the second conservative region C2 of gene env, so far unknown, was detected and characterized. In HIV-1 the area at the beginning of gene env (5'-env) was found to belong to subtype B and the sequence at the end of gene env (3'-env), to subtype A. The analysis of the amino acid sequence of the third variable region of gene env demonstrated that the viruses under study belonged to macrophagotropic "slow/low" variants, characterized by low replication speed. The analysis of nucleotide sequences of the isolated variants of HIV-1 revealed their close genetic relationship with HIV-1 isolates circulating on the territory of Ukraine.