Altered Protein Conformation Research Articles

Soft Interactions at Nanoparticles Alter Protein Function and Conformation in a Size Dependent Manner

Weak protein-nanoparticle (NP) interactions are studied in a low binding regime as a model for the soft protein corona around nanoparticles in complex biological fluids. Noncovalent, reversible interactions between Subtilisin Carlsberg (SC) and silica NPs shows significant alteration in conformation and enzymatic activity in a NP-size dependent manner. Very weak interactions between SC and silica NPs were revealed by centrifugation-based separations and further supported by small-angle X-ray scattering, while bovine serum albumin was used as a strongly interacting reference. Secondary and tertiary structure changes of SC were studied via circular dichroism and correlated to enzymatic activity where the enzyme kinetics showed a critical role for nanoparticle size.

Arrangement of type IV collagen on NH2 and COOH functionalized surfaces

Apart from the paradigm that cell-biomaterials interaction depends on the adsorption of soluble adhesive proteins we anticipate that upon distinct conditions also other, less soluble ECM proteins such as collagens, associate with the biomaterials interface with consequences for cellular response that might be of significant bioengineering interest. Using atomic force microscopy (AFM) we seek to follow the nanoscale behavior of adsorbed type IV collagen (Col IV)--a unique multifunctional matrix protein involved in the organization of basement membranes (BMs) including vascular ones. We have previously shown that substratum wettability significantly affects Col IV adsorption pattern, and in turn alters endothelial cells interaction. Here we introduce two new model surfaces based on self-assembled monolayers (SAMs), a positively charged -NH(2) , and negatively charged -COOH surface, to learn more about their particular effect on Col IV behavior. AFM studies revealed distinct pattern of Col IV assembly onto the two SAMs resembling different aspects of network-like structure or aggregates (suggesting altered protein conformation). Moreover, the amount of adsorbed FITC-labeled Col IV was quantified and showed about twice more protein on NH(2) substrata. Human umbilical vein endothelial cells attached less efficiently to Col IV adsorbed on negatively charged COOH surface judged by altered cell spreading, focal adhesions formation, and actin cytoskeleton development. Immunofluorescence studies also revealed better Col IV recognition by both α(1) and α(2) integrins on positively charged NH(2) substrata resulting in higher phosphorylated focal adhesion kinase recruitment in the focal adhesion complexes. On COOH surface, no integrin clustering was observed. Taken altogether these results, point to the possibility that combined NH(2) and Col IV functionalization may support endothelization of cardiovascular implants.

Force Generation: ATP-Powered Proteasomes Pull the Rope

Recently, single-molecule force spectroscopy techniques have provided unprecedented opportunities to apply and to quantify forces that guide protein (un-)folding. A new study provides fascinating insights into the sophisticated mechanism by which an ATP-fueled proteolytic machine generates mechanical forces to unfold and translocate multidomain substrates.

Binding studies of phloridzin with human serum albumin and its effect on the conformation of protein

In this study, the binding mode of phloridzin with human serum albumin (HSA) was established under physiological condition. The binding study is important to understand the pharmacokinetics and toxicity of phloridzin. The results proved the mechanism of fluorescence quenching of HSA by phloridzin was due to the formation of HSA–phloridzin complex. The binding constants, the number of binding sites and thermodynamic parameters were calculated. In addition, the alterations of HSA secondary structure in the presence of phloridzin were confirmed by the evidences from Fourier transform infrared (FT-IR), UV–visible absorption, circular dichroism (CD), synchronous and three-dimensional fluorescence spectroscopy. Alterations of protein conformation were observed with reduction of α-helix from 54% (free HSA) to 50% in the HSA–phloridzin complexes, indicating a partial protein unfolding. The distance between phloridzin and HSA was 3.74 nm according to fluorescence resonance energy transfer theory. In addition, the effects of common ions on the constants of HSA–phloridzin complex were also discussed.

Structural and functional analysis of critical amino acids in TMVI of the NHE1 isoform of the Na+/H+ exchanger

The mammalian Na+/H+ exchanger isoform 1 (NHE1) resides on the plasma membrane and exchanges one intracellular H+ for one extracellular Na+. It maintains intracellular pH and regulates cell volume, and cell functions including growth and cell differentiation. Previous structural and functional studies on TMVI revealed several amino acids that are potentially pore lining. We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino acids were highly sensitive to substitution and changes often lead to a dysfunctional protein. Mutations of Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp; Glu248→Gln yielded significant reduction in NHE1 activity. Mutants of Asn227 demonstrated defects in protein expression, targeting and activity. Substituting Asn227→Arg and Ile233→Ala decreased the surface localization and expression of NHE1 respectively. The pore lining amino acids Ile233 and Leu243 were both essential for activity. Glu247 was not essential, but the size of the residue at this location was important while the charge on residue Glu248 was more critical to NHE1 function. Limited trypsin digestion on Leu243→Ala and Glu248→Gln revealed that they had increased susceptibility to proteolytic attack, indicating an alteration in protein conformation. Modeling of TMVI with TMXI suggests that these TM segments form part of the critical fold of NHE1 with Ile233 and Leu465 of TMXI forming a critical part of the extracellular facing ion conductance pathway.

Binding of biogenic and synthetic polyamines to β-lactoglobulin

The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV–vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine–protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of K spm–β-LG = 3.2(±0.6) × 10 4 M −1, K spmd–β-LG = 1.8(±0.5) × 10 4 M −1, K BE-333–β-LG = 5.8(±0.3) × 10 4 M −1 and K BE-3333–β-LG = 6.2(±0.05) × 10 4 M −1. Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine–protein binding affinity: BE-3333 > BE-333 > spermine > spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55–51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine–β-LG complexes, indicating a partial protein unfolding.

Interaction of the Anticancer Plant Alkaloid Sanguinarine with Bovine Serum Albumin

BackgroundInteraction of the iminium and alkanolamine forms of sanguinarine with bovine serum albumin (BSA) was characterized by spectroscopic and calorimetric techniques.Methodology/Principal FindingsFormation of strong complexes of BSA with both iminium and alkanolamine forms was revealed from fluorescence quenching of sanguinarine. Binding parameters calculated from Stern-Volmer quenching method revealed that the neutral alkanolamine had higher affinity to BSA compared to the charged iminium form. Specific binding distances of 3.37 and 2.38 nm between Trp 212 (donor) and iminium and alkanolamine forms (acceptor), respectively, were obtained from Forster resonance energy transfer studies. Competitive binding using the site markers warfarin and ibuprofen, having definite binding sites, demonstrated that both forms of sanguinarine bind to site I (subdomain IIA) on BSA. Sanguinarine binding alters protein conformation by reducing the α-helical organization and increasing the coiled structure, indicating a small but definitive partial unfolding of the protein. Thermodynamic parameters evaluated from isothermal titration calorimetry suggested that the binding was enthalpy driven for the iminium form but favoured by negative enthalpy and strong favourable entropy contributions for the alkanolamine form, revealing the involvement of different molecular forces in the complexation.Conclusions/SignificanceThe results suggest that the neutral alkanolamine form binds to the protein more favourably compared to the charged iminium, in stark contrast to the reported DNA binding preference of sanguinarine.

Analysis of CYP3A4 genetic polymorphisms in Han Chinese

Our study aimed to comprehensively investigate the genetic polymorphisms of CYP3A4 in Han Chinese. We sequenced the gene regions of CYP3A4, including its promoter, exons, surrounding introns and 3' untranslated region (3'UTR), from 100 unrelated-healthy Han Chinese individuals. We detected 11 SNPs, three of which are novel. According to in silico functional prediction of novel variants, 20148 A>G in exon 10, resulting in substitution of Tyr319 with Cys (CYP3A4*21), may induce dramatic alteration of protein conformation, and 26908 G>A in 3'UTR may disrupt post-transcriptional regulation. We identified five alleles in Han Chinese, the allele frequencies of CYP3A4*1, *5, *6, *18 and *21 are 97, 0.5, 1, 1 and 0.5%, respectively. Haplotype inference revealed 14 haplotypes, of which the major haplotype CYP3A4*1A constitutes 59% of the total chromosomes. We also examined the possible role of natural selection in shaping the variation of CYP3A4 and confirmed a trend, consistent with the action of positive selection. We systematically screened the genetic polymorphisms of CYP3A4 in Han Chinese, highlighted possible functional impairment of the novel allele and summarized the distinct allele and haplotype frequency distribution, with an emphasis on detecting the footprint of recent positive selection on the CYP3A4 gene in Han Chinese.

Isoflurane's Effect on Protein Conformation as a Proposed Mechanism for Preconditioning

Persistent alteration of protein conformation due to interaction with isoflurane may be a novel molecular aspect of preconditioning. We preincubated human serum albumin with isoflurane, dialyzed to release agent, and assessed protein conformation. Susceptibility to chemical modification by methylglyoxal and nitrophenylacetate was also examined. Isoflurane had a persistent effect on protein conformation. An increase in the susceptibility of surface residues to chemical modification attended this change in conformation. Modification of isoflurane-treated HSA included intra- and intersubunit cross-linking that may be a consequence of anesthetic-induced changes in multimeric subpopulations. This irreversible effect of isoflurane may represent a mechanism for preconditioning.

ELISA assays and alcohol: increasing carbon chain length can interfere with detection of cytokines

Enzyme-linked immunosorbent assays (ELISAs) are frequently used in studies on cytokine production in response to treatment of cell cultures or laboratory animals. When an ELISA assay is performed on cell culture supernatants, samples often contain the treatment agents. The purpose of the present study was to determine if some of the agents evaluated might inhibit cytokine detection by interfering with the ELISA, leaving the question of whether cytokine production was inhibited unanswered. Mouse and human cytokine ELISA kits from BD Biosciences were used according to the manufacturer’s instructions. Cytokine proteins were subjected to one to five carbon alcohols at 86.8 mM (methanol, ethanol, 1-propanol, 2-propanol, n-butanol, and n-pentanol). After treating cell cultures with alcohols of different carbon chain lengths, we found that some of the alcohols interfered with measurement of some cytokines by ELISA, thus making their effects on cytokine production by cells in culture unclear. Increasing carbon chain length of straight chain alcohols positively correlated with their ability to inhibit detection of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10), but not with the detection of interleukin 6 (IL-6), interleukin 8, (IL-8), and interleukin 12 (IL-12). To avoid misinterpretation of treatment effects, ELISA assays should be tested with the reference protein and the treatment agent first, before testing biological samples. These results along with other recent results we obtained using circular dichroism indicate that alcohols with two or more carbons can directly alter protein conformation enough to disrupt binding in an ELISA (shown in the present study) or to inhibit ligand-induced conformational changes (results not shown). Such direct effects have not been given enough consideration as a mechanism of ethanol action in the immune system.

ADAMs and protein disulfide isomerase: the key to regulated cell-surface protein ectodomain shedding?

The ADAM disintegrin metalloproteinases (where ADAM is 'a disintegrin and metalloproteinase') are a family of transmembrane cell-surface proteins with essential roles in adhesion and proteolytic processing in all animals. The archetypal family member is ADAM17 {also known as TACE [TNFalpha (tumour necrosis factor alpha)-converting enzyme]}, which is involved in processing pro-TNFalpha and in the activation of ligands for the EGFR [EGF (epidermal growth factor) receptor], as well as cleavage of diverse cell-surface receptors and adhesion molecules. ADAM-mediated shedding is itself influenced via cell signalling pathways. In this issue of the Biochemical Journal, Willems et al. make the observation that phorbol ester activates shedding by ADAM17 by affecting the activity of PDI (protein disulfide isomerase). They propose that PDI maintains ADAM17 in an inactive 'closed' state and PMA stimulation generates ROS (reactive oxygen species) and thus an altered redox environment, which in turn inactivates PDI and allows ADAM17 to adopt an 'open' active conformation. This activation is accompanied by changes in disulfide bonds in the ADAM17 ectodomain. This is a novel and exciting finding that could help to unlock the actions of ADAM sheddases, as well as a host of other mechanisms that rely upon rapid alterations in protein conformation on the cell surface.

Biogenic and Synthetic Polyamines Bind Bovine Serum Albumin

Biogenic polyamines are found to modulate protein synthesis at different levels, while polyamine analogues have shown major antitumor activity in multiple experimental models, including breast cancer. The aim of this study was to examine the interaction of bovine serum albumin (BSA) with biogenic polyamines, spermine and spermidine, and polyamine analogues 3,7,11,15-tetrazaheptadecane x 4 HCl (BE-333) and 3,7,11,15,19-pentazahenicosane x 5 HCl (BE-3333) in aqueous solution at physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind BSA via both hydrophilic and hydrophobic interactions. Stronger polyamine-protein complexes formed with biogenic than synthetic polyamines with overall binding constants of K(spm) = 3.56 (+/-0.5) x 10(5) M(-1), K(spmd) = 1.77 (+/-0.4) x 10(5) M(-1), K(BE-333) = 1.11 (+/-0.3) x 10(4) M(-1) and K(BE-3333) = 3.90 (+/-0.7) x 10(4) M(-1) that correlate with their positively charged amino group contents. Major alterations of protein conformation were observed with reduction of alpha-helix from 63% (free protein) to 55-33% and increase of turn 12% (free protein) to 28-16% and random coil from 6% (free protein) to 24-17% in the polyamine-BSA complexes, indicating a partial protein unfolding. These data suggest that serum albumins might act as polyamine carrier proteins in delivering polyamine analogues to target tissues.

Water structure theory and some implications for drug design.

The development of theories of water structure has been hindered in the past by the difficulty of experimental measurement. Both measurement and computer modelling studies have now reached the stage where theoretical treatments of water structure are converging to a broadly acceptable model. In current understanding, water is a mixture of randomly hydrogen-bonded molecules and larger structures comprised of tetrahedral oxygen centres which, when hydrogen-bonded to each other, lead to five-membered and other rings which can aggregate to form three-dimensional structures. Evidence is taken from studies of the ices, from clathrates and other solid solutions, as well as from liquid solutions, that certain motifs occur very frequently and have relatively high stability, such as the (H2O)20 cavity-forming structure known from studies on clathrates. The implications of recent models of water structure for an understanding of biological events, including the interactions of drugs with receptors, are profound. It is becoming clear that modelling of aqueous solutions of any molecule must consider the explicit interactions with water molecules, which should not be regarded as a continuum: water itself is not a continuum. Solute molecules which possess hydrogen-bonding groups will provoke the formation of further hydrogen-bonding chains of water molecules: if these can form rings, such ringswilltend to persist longerthan chains, givingthesolute a secondary identity of associated water which may play a role in molecular recognition. Solutes that do not have hydrogen-bonding capability, or regions of solutes which are non-polar, may also produce partial cage-like water structures that are characteristic of the solute. The classification of many solutes as structure makers or structure breakers has relevance to the interactions between ligands and large biomolecules such as proteins. While it is generally accepted that sulfate and urea, respectively structure maker and breaker, may alter protein conformation through effects on water, it has not been recognised that bioactive ligands, which also change the conformation of proteins, may do so by a related, but more selective, mechanism. Very early studies of cell contents suggested that the associated water might be different from bulk water, a concept that lost support in the mid-20th century. Current theories of water structure may invite a reappraisal of this position, given the observation that structuring may extend for many molecular diameters from an ordered surface.

How do bacteria sense and respond to low temperature?

Rigidification of the membrane appears to be the primary signal perceived by a bacterium when exposed to low temperature. The perception and transduction of the signal then occurs through a two-component signal transduction pathway consisting of a membrane-associated sensor and a cytoplasmic response regulator and as a consequence a set of cold-regulated genes are activated. In addition, changes in DNA topology due to change in temperature may also trigger cold-responsive mechanisms. Inducible proteins thus accumulated repair the damage caused by cold stress. For example, the fluidity of the rigidified membrane is restored by altering the levels of saturated and unsaturated fatty acids, by altering the fatty acid chain length, by changing the proportion of cis to trans fatty acids and by changing the proportion of anteiso to iso fatty acids. Bacteria could also achieve membrane fluidity changes by altering the protein content of the membrane and by altering the levels of the type of carotenoids synthesized. Changes in RNA secondary structure, changes in translation and alteration in protein conformation could also act as temperature sensors. This review highlights the various strategies by which bacteria senses low temperature signal and as to how it responds to the change.

NMR analysis of G-protein βγ subunit complexes reveals a dynamic Gα-Gβγ subunit interface and multiple protein recognition modes

G-protein betagamma (Gbetagamma) subunits interact with a wide range of molecular partners including: G(alpha) subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gbetagamma, a method for site-specific (15)N-labeling of Gbeta-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of (15)N-Trp Gbetagamma identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GbetaW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GbetaW99 is highly mobile on the nanosecond timescale in comparison with other Gbeta tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GbetaW99 and GbetaW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of G(alpha)(i1)-GDP to Gbetagamma had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gbetagamma subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.

The effect of methylamine on the solution structures of human and bovine serum albumins

Serum albumins are the major soluble protein constituents of the circulatory system and have many physiological functions including transporting a variety of compounds. Methylamine, a monoamine with one positive charge complexes with protein and alters protein secondary structure. The aim of this study was to examine the interactions of human serum albumin (HSA) and bovine serum albumin (BSA) with methylamine at physiological conditions, using constant protein concentration and various monoamine concentrations. FTIR, UV–vis, CD and fluorescence spectroscopic methods were used to analyse methylamine binding mode, the binding constant and the effects of monoamine on HSA and BSA stability and conformations. Structural analysis showed that methylamine binds HSA and BSA via hydrophilic (polypeptide and amine polar groups) and hydrophobic interactions with overall binding constants of K met-HSA = 2.42 (±0.5) × 10 2 M −1 and K met-BSA = 1.34 (±0.3) × 10 3 M −1 with the number of bound methylamine around one molecule per protein. Methylamine complexation alters protein conformation by major reduction of α-helix and increase in random coil and turn structures indicating a partial protein unfolding.

Exploiting fluorescence resonance energy transfer to probe structural changes in a macromolecule during adsorption and incorporation into a growing biomineral crystal

The growth of natural biominerals is often tightly regulated by surface adsorption and subsequent incorporation of proteins into the crystal structure. Understanding how macromolecules intercalate into inorganic crystal lattices and how incorporation affects protein structure is crucial to learning how to engineer biomimetic materials with advanced properties, yet knowledge about the molecular-level interactions between organic guests and inorganic hosts remains sparse. Here we have used fluorescence resonance energy transfer (FRET) to probe conformational changes of a macromolecule as it adsorbs to, and becomes incorporated within, a biomineral crystal. Calcium oxalate monohydrate (COM) was used as a model due to its large size and kinetic stability under a wide range of pH values. Since the conformation of the extracellular matrix protein fibronectin (Fn) is highly sensitive to local ion concentrations, major conformational changes can be observed by FRET, as Fn senses and responds to varying local ionic conditions. When transferred from a physiological buffer to a supersaturated solution, Fn's crossed-over dimeric arms separate, indicating a weakening of the electrostatic interactions which otherwise stabilize the compact conformation of the protein. Fn returns to a more compact state when binding to the flat (−1 0 1) surface of the crystal, suggesting that Fn might sense a zone of ion depletion right at the interface of the growing crystal. As the crystal begins to grow around the absorbed protein, the dimeric Fn arms separate again, potentially driven by interactions with the newly formed charged step edges forming around it during the embedding process. FRET thus reveals for the first time how local changes in the electrostatic environment during the growth of a biomineral can cause major alterations in protein conformation. The insights derived using FRET and atomic force microscopy (AFM) could stimulate novel ways to tailor and tune the properties of organic–inorganic composites by exploiting dynamically changing electrostatic guest–host interactions.

Ultrafast Protein Conformational Alterations in Bacteriorhodopsin and Its Locked Analogue BR5.12

Bacteriorhodopsin, reconstituted with a sterically "locked" retinal chromophore, BR5.12, has frequently been used to elucidate elementary photoinduced processes in the native pigment bacteriorhodopsin. In this work, the vibrational response of BR5.12 to photoexcitation is investigated by means of femtosecond time-resolved mid-infrared and UV-vis spectroscopy. The electronically excited state of BR5.12 decays with a time constant of 18 ps. Neither in the UV-vis nor in the mid-IR spectral region are indications found for chromophore photoproducts, besides the full recovery of the electronic ground state. However, vibrational bands are observed at around 1660 and 1550 cm(-1) in the protein amide I and amide II band regions, respectively. They are formed within a few picoseconds or even instantaneously. Thus, they appear faster than the S1 decay and persist for at least 130 ps, i.e., for much longer than the S1 lifetime. These findings strongly suggest that the observed bands must be assigned to protein vibrations and that they are not caused by a photoinduced temperature rise. Thus, for the first time, ultrafast protein vibrational changes are detected in BR5.12, that are not associated with isomerization. Possibly they can be related to the enhanced chemical reactivity of photoactivated BR5.12 reported in the literature. In wild-type bacteriorhodopsin, bands with very similar spectral and kinetic characteristics are observed, suggesting that they might originate from a similar mechanism which is not isomerization. A plausible mechanism is a polarization induced protein conformational change, as discussed in the literature.

The EG95 Antigen of Echinococcus spp. Contains Positively Selected Amino Acids, which May Influence Host Specificity and Vaccine Efficacy

Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.

Acute Alcohol Action and Desensitization of Ligand-Gated Ion Channels

Ethanol exerts its biological actions through multiple receptors, including ion channels. Ion channels that are sensitive to pharmacologically relevant ethanol concentrations constitute a heterogeneous set, including structurally unrelated proteins solely sharing the property that their gating is regulated by a ligand(s). Receptor desensitization is almost universal among these channels, and its modulation by ethanol may be a crucial aspect of alcohol pharmacology and effects in the body. We review the evidence documenting interactions between ethanol and ionotropic receptor desensitization, and the contribution of this interaction to overall ethanol action on channel function. In some cases, such as type 3 serotonin, nicotinic acetylcholine, GABA-A, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors, ethanol actions on apparent desensitization play a significant role in acute drug action on receptor function. In a few cases, mutagenesis helped to identify different areas within a receptor protein that differentially sense n-alcohols, resulting in differential modulation of receptor desensitization. However, desensitization of a receptor is linked to a variety of biochemical processes that may alter protein conformation, such as the lipid microenvironment, post-translational channel modification, and channel subunit composition, the relative contribution of these processes to ethanol interactions with channel desensitization remains unclear. Understanding interactions between ethanol and ionotropic receptor desensitization may help to explain different ethanol actions 1) when ethanol is evaluated in vitro on cloned channel proteins, 2) under physiological or pathological conditions or in distinct cell domains with modified ligand concentration and/or receptor conformation. Finally, receptor desensitization is likely to participate in molecular and, possibly, behavioral tolerance to ethanol, which is thought to contribute to the risk of alcoholism.