Molecular mechanisms of α-synuclein neurodegeneration

α-Synuclein is an abundant highly charged protein that is normally predominantly localized around synaptic vesicles in presynaptic terminals. Although the function of this protein is still ill-defined, genetic studies have demonstrated that point mutations or genetic alteration (duplications or triplications) that increase the number of copies of the α-synuclein (SCNA) gene can cause Parkinson's disease or the related disorder dementia with Lewy bodies. α-Synuclein can aberrantly polymerize into fibrils with typical amyloid properties, and these fibrils are the major component of many types of pathological inclusions, including Lewy bodies, which are associated with neurodegenerative diseases, such as Parkinson's disease. Although there is substantial evidence supporting the toxic nature of α-synuclein inclusions, other modes of toxicity such as oligomers have been proposed. In this review, some of the evidence for the different mechanisms of α-synuclein toxicity is presented and discussed.     

Voltage-activated complexation of α-synuclein with three diverse β-barrel channels: VDAC,MspA, and α-hemolysin

Voltage-activated complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or α-synuclein (αSyn). However, the roles played by VDAC's characteristic attributes—its anion selectivity and voltage gating behavior—have remained unclear. Here, we compare in vitro measurements of voltage-activated complexation of αSyn with three well-characterized β-barrel channels—VDAC, MspA, and α-hemolysin—that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels are observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play a role. The reaction free energy landscapes thus calculated reveal a non-trivial dependence of the αSyn/channel complex stability on the surface density of αSyn.     

Molecular Divergence of Lysozymes and α-Lactalbumin

Abstract

The vast number of proteins that sustain the currently living organisms have been generated from a relatively small number of ancestral genes that has involved a variety of processes. Lysozyme is an ancient protein whose origin goes back an estimated 400 to 600 million years. This protein was originally a bacteriolytic defensive agent and has been adapted to serve a digestive function on at least two occasions, separated by nearly 40 million years. The origins of the related goose type and T4 phage lysozyme that are distinct from the more common C type are obscure. They share no discernable amino acid sequence identity and yet they possess common secondary and tertiary structures. Lysozyme C gene also gave rise, after gene duplication 300 to 400 million years ago, to a gene that currently codes for α-lactalbumin, a protein expressed only in the lactating mammary gland of all but a few species of mammals. It is required for the synthesis of lactose, the sugar secreted in milk. α-Lactalbumin shares only 40% identity in amino acid sequence with lysozyme C, but it has a closer spatial structure and gene organization. Although structurally similar, functionally they are quite distinct. Specific amino acid substitutions in α-lactalbumin account for the loss of the enzyme activity of lysozyme and the acquisition of the features necessary for its role in lactose synthesis. Evolutionary implications are as yet unclear but are being unraveled in many laboratories.     

A Molecular Trajectory of α-Actinin Activation

The mechanisms by which living cells respond to mechanical stimuli are not yet fully understood. It has been suggested that mechanosensing proteins play an important role in mechanotransduction because their binding affinities are directly affected by the external stress. α-Actinin is an actin cross-linker and may act as a mechanosensor in adhesion sites. Its interaction with vinculin is suggested to be mechanically regulated. In this study, the free energy of activation is explored using the umbrella sampling method. An activation trajectory is generated in which α-actinin’s vinculin-binding site swings out of the rod domain, leading to approximately an 8 kcal/mol free energy release. The activation trajectory reveals several local and global conformational changes along the activation pathway accompanied by the breakage of a number of key interactions stabilizing the inhibited structure. These results may shed light on the role of α-actinin in cellular mechanotransduction and focal adhesion formation.     

Molecular basis of α-thalassemia in Sicily

To evaluate the allelic frequency and genetic diversity of α-thalassemia defects in Sicily, both epidemiological and patient-oriented studies were carried out. For the epidemiological study, phenotypic data were collected on more than 1000 Sicilian individuals. Among them, 427 were explored at the molecular level for nine α-thalassemic variants known to be common in the Mediterranean region. Our data reveal an allele frequency of 4.1% for α⁺-thalassemia matching that of β-thalassemia in this region. The presence of α°-thalassemia (––^MEDI and ––^CAL) was observed only in the group of referred patients. Newly acquired nucleotide sequence data on the deletional breakpoint of ––^CAL allowed us to design a simple PCR-based procedure for exploring this allele. The data also provide additional information concerning the genetic mechanisms involved in such large deletions. Received: 8 August 1996 / Revised: 16 October 1996     

Molecular Modeling and Physicochemical Properties of Supramolecular Complexes of Limonene with α- and β-Cyclodextrins

This study evaluated three different methods for the formation of an inclusion complex between alpha- and beta-cyclodextrin (α- and β-CD) and limonene (LIM) with the goal of improving the physicochemical properties of limonene. The study samples were prepared through physical mixing (PM), paste complexation (PC), and slurry complexation (SC) methods in the molar ratio of 1:1 (cyclodextrin:limonene). The complexes prepared were evaluated with thermogravimetry/derivate thermogravimetry, infrared spectroscopy, X-ray diffraction, complexation efficiency through gas chromatography/mass spectrometry analyses, molecular modeling, and nuclear magnetic resonance. The results showed that the physical mixing procedure did not produce complexation, but the paste and slurry methods produced inclusion complexes, which demonstrated interactions outside of the cavity of the CDs. However, the paste obtained with β-cyclodextrin did not demonstrate complexation in the gas chromatographic technique because, after extraction, most of the limonene was either surface-adsorbed by β-cyclodextrin or volatilized during the procedure. We conclude that paste complexation and slurry complexation are effective and economic methods to improve the physicochemical character of limonene and could have important applications in pharmacological activities in terms of an increase in solubility.     

Molecular quantification of Saccharomyces cerevisiae α-pheromone secretion

Saccharomyces cerevisiae yeast cells court each other by producing an attractive sex pheromone specific to their mating type. Cells detect the sex pheromone from potential mates using a well-defined intracellular signalling cascade that has become a model for studying signal transduction. In contrast, the factors contributing to the production of pheromone itself are poorly characterized, despite the widespread use of the S.?cerevisiae α-pheromone secretion pathway in industrial fungal protein expression systems. Progress in understanding pheromone secretion has been hindered by a lack of a precise and quantitative pheromone production assay. Here, we present an ELISA-based method for the quantification of α-pheromone secretion. In the absence of pheromone from the opposite mating type, we found that each cell secretes over 550 mature α-pheromone peptides per second; 90% of this total was produced from MF α1. The addition of a-pheromone more than doubled total α-pheromone secretion. This technique offers several improvements on current methods for measuring α-pheromone production and will allow detailed investigation of the factors regulating pheromone production in yeast.     

Increased yield of biotransformation of exemestane with β-cyclodextrin complexation technique

In this study, 6-methylenandrosta-4-ene-3,17-dione and Hydroxypropyl-β-cyclodextrin (HP-β-CD) were used to form a complex, which could be then biotransformed by Arthrobacter simplex ATCC6946 to obtain the antitumor drug exemestane. The complex was analyzed by UV, DSC and TG techniques, while the products were analyzed by HPLC, NMR and MS. These results confirmed that the β-cyclodextrin not only improved the water-solubility of 6-methylenandrosta-4-ene-3,17-dione, but also greatly enhanced the biocompatibility during the biotransformation process. This result may be applied to other precursors which have poor aqueous solubility in the biotransformation processes.     

Enhancement of naringenin bioavailability by complexation with hydroxypropoyl-β-cyclodextrin

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with β-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-β-cyclodextrin (HPβCD), specifically, increased the solubility of naringenin by over 400-fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPβCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and C(max) increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1α in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPβCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.     

Molecular dynamics study of an α-cyclodextrin-phosphatidylinositol inclusion complex

Cyclodextrins are cyclic oligosaccharides known for their ability to include substrate molecules in their hydrophobic cavity. Moreover, cyclodextrins show a hemolytic activity when mm concentrations are added to blood. This hemolysis is commonly interpreted as a massive dissociation of phospholipids from the cell membrane due to the formation of complexes with the cyclodextrins. In the literature, a complexation between -cyclodextrin ( CD) and phosphatidylinositol (PI) specific to the inositol headgroup has been proposed. But the need for the detailed interaction mechanism between the two molecules motivated the present work based on molecular dynamics simulations. Investigation of long range electrostatic interactions shows that a mutual approach of the molecules is only possible when the primary hydroxyl side of CD faces the inositol headgroup of PI. This orientation is also the most favourable from adiabatic- and free-energy profiles calculated along a reaction coordinate that leads to an inclusion of PI into a CD. For free energy simulations, partial hydration of the model has been used. A study of glycosidic bond dihedral angles in CD shows an increase in dihedral fluctuations before complexation and a dihedral freezing once the complex is formed.     

Molecular chaperone α-crystallin prevents detrimental effects of neuroinflammation

Silver nitrate administration stimulates immune activation, inflammation and deterioration in cell function. It is well established that hippocampal and cortical tissue are susceptible to degeneration in responses to insult such as oxidative stress or infection. This study was designed to investigate the prophylactic effect of α-crystallin, a major chaperone lens protein comprising of α-A and α-B subunits in inflammation induced mice. Mice were divided into three groups (n = 6 in each), control, inflammation and α-crystallin treated. Our result shows that α-crystallin pretreatment effectively diminished systemic inflammation induced glial fibrillary acidic protein (GFAP) and nuclear factor kappa B (NFκB) expression in the mice neocortex, reversed elevated intracellular calcium levels, acetylcholine esterase activity and depletion of glucose. Furthermore it also significantly prevented nitric oxide (P < 0.05) and lipid peroxide production in the plasma, liver, neocortex and hippocampus of the inflammation-induced mice. In order to demonstrate the direct OH and nitric oxide radical scavenging ability of α-crystallin, an In vitro experiment using primary astrocyte culture subjected to lipopolysaccharide (LPS), a well-known inflammatory stimuli were also carried out. This study reiterates that α-crystallin therapy may serve as a potent pharmacological agent in neuroinflammation.     

Molecular modeling of the intercalation of porphyrins into α-zirconium phosphate

In this work, n-alkylamines (number of carbon atoms ranging from 3 to 10) were investigated in detail by molecular modeling as spacers for intercalating porphyrins into α-zirconium phosphate (α-ZrP). Pre-intercalated n-alkylamines can form either a flat monolayer or a canted bilayer in the gallery of α-ZrP. Based on the interlayer state and intercalative potential of the two modes in α-ZrP, it is suggested that the flat monolayer is a better spacer than the bilayer and that n-propylamine (PA) and n-butylamine (BA) in mobile monolayers are the best spacers among the n-alkylamines studied, as is also found experimentally. The intercalation behavior of TMPyP [5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin] and several other porphyrins was investigated by calculating the intercalative potential. The calculated results showed that the porphyrins were densely packed in a canted monolayer model, and an increase of polarity of the substituent would facilitate the intercalation of the porphyrins. Figure Schematic representation of platform of intercalated spacers and guests taking n-butylamine and TMPyP as an example, respectively: a a flat monolayer of n-bultylamine in α-ZrP; b a canted monolayer of TMPyP in α-ZrP; c the top layer of the canted bilayer n-bultylamine in α-ZrP (the gray area indicates the amphiphilic distribution on the interface between α-ZrP layers and n-alkylamine/porphyrin).      

In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.     

Influence of hydrophilic polymers on celecoxib complexation with hydroxypropyl β-cyclodextrin

Complexation of celecoxib with hydroxypropyl β-cyclodextrin (HPβCD) in the presence and absence of 3 hydrophilic polymers—polyvinyl pyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), and polyethylene glycol (PEG)—was investigated with an objective of evaluating the effect of hydrophilic polymers on the complexation and solubilizing efficiencies of HPβCD and on the dissolution rate of celecoxib from the HPβCD complexes. The phase solubility studies indicated the formation of celecoxib-HPβCD inclusion complexes at a 1∶1M ratio in solution in both the presence and the absence of hydrophilic polymers. The complexes formed were quite stable. Addition of hydrophilic polymers markedly enhanced the complexation and solubilizing efficiencies of HPβCD. Solid inclusion complexes of celecoxib-HPβCD were prepared in 1∶1 and 1∶2 ratios by the kneading method, with and without the addition of hydrophilic polymers. The solubility and dissolution rate of celecoxib were significantly improved by complexation with HPβCD. The celecoxib-HPβCD (1∶2) inclusion complex yielded a 36.57-fold increase in the dissolution rate of celecoxib. The addition of hydrophilic polymers also markedly enhanced the dissolution rate of celecoxib from HPβCD complexes: a 72.60-, 61.25-, and 39.15-fold increase was observed with PVP, HPMC, and PEG, respectively. Differential scanning calorimetry and X-ray diffractometry indicated stronger drug amorphization and entrapment in HPβCD because of the combined action of HPβCD and the hydrophilic polymers. Published: September 29, 2006     

New Molecular Determinant for Inactivation of the Human L-Type α1C Ca2+ Channel

Molecular cloning of the human fibroblast Ca²⁺ channel pore-forming α_1C subunit revealed (Soldatov, 1992. Proc. Natl. Acad. Sci. USA 89:4628-4632) a naturally occurring mutation g²²⁵⁴→ a that causes the replacement of the conservative alanine for threonine at the position 752 at the cytoplasmic end of transmembrane segment IIS6. Using stably transfected HEK293 cell lines, we have compared electrophysiological properties of the conventional α_1C,77 human recombinant L-type Ca²⁺ channel with those of its mutated isoform α_1C,94 containing the A752T replacement. Comparative quantification of steady-state availability of the current carried by α_1C,94 and α_1C,77 showed that A752T mutation prevented a large (≈25%) fraction of the current carried by Ca²⁺ or Ba²⁺ from fully inactivating. This mutation, however, did not appear to alter significantly the Ca²⁺-dependence and kinetics of decay of the inactivating fraction of the current or its voltage-dependence. The data suggests that Ala752 at the cytoplasmic end of IIS6 might serve as a molecular determinant of the Ca²⁺ channel inactivation, possibly regulating the voltage-dependence of its availability. Received: 14 January 2000/Revised: 20 June 2000     

Characterization of a α-l-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C

In this study, a α-l-rhamnosidase gene from Bacteroides thetaiotaomicron VPI-5482 was cloned and expressed in Escherichia coli. The specific activity of rhamnosidase was 0.57 U/mg in LB medium with 0.1 mM Isopropyl β-d-Thiogalactoside (IPTG) induction at 28 °C for 8 h. The protein was purified by Ni-NTA affinity, which molecular weight approximately 83.3 kDa. The characterization of BtRha was determined. The optimal activity was at 55 °C and pH 6.5. The enzyme was stable in the pH range 5.0–8.0 for 4 h over 60%, and had a 1-h half-life at 50 °C. The Kcat and Km for p-nitrophenyl-α-l-rhamnopyranoside (pNPR) were 1743.29 s⁻¹ and 2.87 mM, respectively. The α-l-rhamnosidase exhibited high selectivity to cleave the α-1,2 and α-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively, which could hydrolyze rutin, hesperidin, epimedin C and 2″-O-rhamnosyl icariside II. Under the optimal conditions, BtRha transformed epimedin C (1 g/L) to icariin by 90.5% in 4 h. This study provides the first demonstration that the α-l-rhamnosidase could hydrolyze α-1,2 glycosidic bond between rhamnoside and rhamnoside.