The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

The Role of Various Domains of the Iron–Sulfur Protein in the Assembly and Activity of the Cytochromme bc 1 Complex of Yeast Mitochondria

Assembly studies in vitro of deletion mutants of the iron–sulfur protein into the cytochromebc ₁ complex revealed that mutants localized in the extramembranous regions of the proteinwere not assembled into the complex in contrast to the efficient assembly of mutants in themembrane-spanning region. Charged amino acids located in the extramembranous 1-4 loopand the 1 helix were mutated and expressed in yeast cells lacking the gene for the iron–sulfurprotein. Mutating the charged amino acid residues H124, E125, R146, K148, and D149 aswell as V132 and W152 resulted in loss of enzymatic activity due to the loss of iron–sulfurprotein suggesting that these amino acids are required to maintain protein stability. By contrast,no loss of iron–sulfur protein accompanied the 30–50% loss of bc ₁ complex activity in mutantsof three conserved alanine residues, A86, A90, and A92, suggesting that these residues maybe involved in the proposed movement of the flexible tether of the iron–sulfur proteinduring catalysis.     

Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of αA- and αB-crystallins

Earlier studies have shown significant loss of chaperone activity in α-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of α-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in α-crystallin have been identified as the major glycation sites: K11, K78, and K166 in αA-crystallin and K90, K92, and K166 in αB-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of αA showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to αA-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of αB showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. αA-K11T and αB-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.     

Role of Ferrocyanides in the Prebiotic Synthesis of α-Amino Acids

We investigated the synthesis of α-amino acids under possible prebiotic terrestrial conditions in the presence of dissolved iron (II) in a simulated prebiotic ocean. An aerosol-liquid cycle with a prebiotic atmosphere is shown to produce amino acids via Strecker synthesis with relatively high yields. However, in the presence of iron, the HCN was captured in the form of a ferrocyanide, partially inhibiting the formation of amino acids. We showed how HCN captured as Prussian Blue (or another complex compound) may, in turn, have served as the HCN source when exposed to UV radiation, allowing for the sustained production of amino acids in conjunction with the production of oxyhydroxides that precipitate as by-products. We conclude that ferrocyanides and related compounds may have played a significant role as intermediate products in the prebiotic formation of amino acids and oxyhydroxides, such as those that are found in iron-containing soils and that the aerosol cycle of the primitive ocean may have enhanced the yield of the amino acid production.     

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65^−/− mouse model of Leber''s congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.     

Mitochondrial Ageing and the Beneficial Role of α-Lipoic Acid

Oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the ageing process. Mitochondria are known to be a rich source for the production of free radicals and, consequently, mitochondrial components are susceptible to lipid peroxidation (LPO) that decreases respiratory activity. In the present investigation, we have evaluated mitochondrial LPO, 8-oxo-dG, oxidized glutathione, reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and electron transport chain (ETC) complex activities in the brain of young versus aged rats. In aged rats, the contents of LPO, oxidized glutathione and 8-oxo-dG were high whereas reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities were found to be low. Lipoic acid administration to aged rats reduced the levels of mitochondrial LPO, 8-oxo-dG and oxidized glutathione and enhanced reduced glutathione, ATP, lipoic acid and ETC complex activities. In young rats lipoic acid administration showed only minimal lowering the levels of LPO, 8-oxo-dG and oxidized glutathione and slight increase in the levels of reduced glutathione, ATP, lipoic acid, TCA cycle enzymes and ETC complex activities. These findings suggest that the dithiol, lipoic acid, provides protection against age-related oxidative damage in the mitochondria of aged rats.     

Role of the structural context in selection of hydrophobic side-chain rotamers in a- and d-positions of α-helices

It was demonstrated for the first time that the distribution of side-chain rotamers in the a-and d-positions of α-helices of coiled-coil (cc) proteins follows a certain trend, rather then being random. For instance, most side chains adopt t rotamers in the a-positions and g^? rotamers in the d-positions of helical dimers. Vice versa, most side chains adopt g^? rotamers in the a-positions and t rotamers in the d-positions of tetramers. It was concluded that selection of the side-chain rotamers depends on the packing of α-helices and, consequently, depends on the structural context.     

Dominant-Negative Mutations in the G-Protein-Coupled α-Factor Receptor Map to the Extracellular Ends of the Transmembrane Segments

G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled α-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the α-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.G-protein-coupled receptors (GPCRs) comprise a large family of receptors that are found in a wide range of eukaryotic organisms from yeasts to humans (4, 10). These receptors respond to diverse stimuli including hormones, neurotransmitters, and other chemical messengers (48). GPCRs transduce their signal by stimulating the α subunit of a heterotrimeric guanine nucleotide binding protein (G protein) to bind GTP (4, 16). This releases the α subunit from the βγ subunits, and then either the α subunit or the βγ subunits go on to promote signaling depending on the specific pathway (28).GPCRs are structurally similar in that they contain seven transmembrane domains (TMDs) connected by intracellular and extracellular loops. Although many techniques have been applied to study receptor function, much of our knowledge on the mechanisms of GPCR activation comes from the characterization of mutant receptors. Loss-of-function and supersensitive mutants have helped to identify receptor regions needed for ligand binding, G-protein activation, and down-regulation of signaling (4, 49). Furthermore, the study of constitutively active receptor mutations has played a key role in the development of current models for receptor activation (26). Naturally occurring GPCR mutations have also been implicated in a number of human diseases (8, 25, 42). Interestingly, the analysis of different mutant receptors indicates that GPCRs utilize common structural domains for similar functions. In particular, the third intracellular loop has an essential role in G-protein activation in a wide range of GPCRs.The genetic approaches possible in the yeast Saccharomyces cerevisiae have been used to examine the relationship between structure and function of the G-protein-coupled mating pheromone receptors. The α-factor and a-factor pheromones induce conjugation in yeast by binding to receptors with seven TMDs that activate a G-protein signal pathway that is highly conserved with mammalian signaling pathways (24). In fact, some human GPCRs can activate the pheromone signal pathway when they are expressed in yeast (19, 29). The analysis of loss-of-function, supersensitive, and constitutively active α-factor receptor mutants has begun to reveal the mechanisms for activation and regulation of this receptor. For example, the analysis of constitutively active mutants indicates that movement in the transmembrane segments plays a key role in α-factor receptor activation (22). Constitutive mutations and loss-of-function mutations implicate the third intracellular loop in G-protein activation (7, 34, 44). Mutagenesis studies also indicate that the cytoplasmic C terminus is not needed for G-protein activation but is involved in down-regulation of receptors by endocytosis (17) and desensitization of receptors by phosphorylation (6). In addition, studies with chimeric receptors suggest that the specificity for α-factor binding is determined by discontinuous segments of the α-factor receptor that include the transmembrane and extracellular regions (36, 37). Although some of the important domains of the α-factor receptor have been identified in these studies, the molecular mechanism of receptor signaling remains to be determined.Dominant-negative (DN) mutants represent an important class of mutation in which a mutant receptor interferes with the function of the wild-type (WT) version of the receptor. Since the inhibitory phenotype in DN mutants implies loss of some but not all functions of the protein, these mutants have been used to great advantage in other receptor systems. For example, in the case of receptor tyrosine kinases, DN mutants have been used to assign particular functions to specific structural features or to study the effects of blocking receptor signaling (18). In view of the large number of mutations reported for GPCRs, it is intriguing that there are few examples of dominant GPCR mutations (42, 43). Furthermore, in cases where it has been examined, dominant mutations in GPCRs seem to affect primarily the targeting of receptors to the plasma membrane and not directly the function of the WT receptors. Therefore, we sought to determine if the analysis of DN mutants could be applied to GPCRs by taking advantage of the genetic accessibility of the yeast S. cerevisiae. In this report, we describe the identification of DN mutations in the α-factor pheromone receptor. Interestingly, our results indicate that these DN mutants interfere with the activity of the WT receptors by competing for the G protein. In addition, these mutations identify a new domain on the extracellular side of the TMDs that is important for receptor function.     

Hydroxylysine in the N-terminal regions of the α1- and α2-chains of various collagens

The degree of hydroxylation of the lysine residue located in both alpha(1)- and alpha(2)-chains of collagen in the N-terminal, non-helical telopeptide region of the molecule has been determined in collagen from various sources after isolation of the peptides (alpha(1)- and alpha(2)-CB1) that contain the lysine residue in question and are obtained by cyanogen bromide cleavage of collagen alpha(1)- and alpha(2)-chains respectively. As with collagen from chick tibia, bone collagens from rat tibia and femur and embryonic chick frontal bone, have a high degree of hydroxylation (approx. 50% or more) of the lysine residue in both alpha(1)- and alpha(2)-CB1 peptides. This is in contrast with the lack of hydroxylation of this residue in both alpha(1)- and alpha(2)-chains of all skin collagens so far examined. The presence of hydroxylysine in alpha(1)- and alpha(2)-CB1 peptides from tendon collagen is also indicated. In rat tail tendon collagen the amount of hydroxylation is only slight but in the much less soluble tendon collagen from embryonic chick leg tendons, approximately one-third of the lysine is hydroxylated.     

Role of A-chain in functioning of the active site of human α-thrombin

This review summarizes current data suggesting that A-chain of the human alpha-thrombin molecule plays a role of allosteric effector in catalytic reactions with various substrates. Special attention is paid to the relationship between A-chain structure and catalytic activity of thrombin. The existence of this relationship is based on studies of natural mutation of A-chain of the alpha-thrombin molecule. Use of molecular and essential dynamics confirmed the role of A-chain in changes of conformation and catalytic properties of this enzyme; these changes involve residues located in the specificity sites and some inserting loops. Current knowledge on structure and properties of thrombin can be used for the development of new antithrombin agents.     

α1-adrenoceptors in neonatal rat cardiac myocytes: Hypoxia alters the responsiveness of α1A and α1B subtypes

We investigated the contribution of α₁-adrenoceptor subtypes to the chronotropic response to norepinephrine (NE) in cultured neonatal rat cardiac myocytes under normoxia and hypoxia. A dose-dependent negative chronotropic response was induced by NE in the presence of propranolol. Hypoxic exposure inverted the negative chronotropic response to NE to a positive one. All of these chronotropic responses were completely antagonized by prazosin. In normoxic conditions, the NE-induced negative chronotropic response was completely antagonized by WB4101 but only partially (55%) so by chloroethylclonidine (CEC). After hypoxic exposure, WB4101 partially antagonized the positive chronotropic response to NE (54%), while CEC completely suppressed the action of NE. Hypoxic exposure did not alter the number of α_1A - and α_1B-adrenoceptor subtypes as measured by [³H]prazosin binding following CEC treatment. These results indicate (1) that cultured neonatal rat cardiac myocytes contain both α₁-adrenoceptor subtypes, i.e., α_1A and α_1B, and (2) that the predominant α₁-adrenoceptor subtypes mediating NE-induced chronotropy were altered by hypoxia.     

Molecular Analysis of the α-Galactosidase MEL Genes in Yeast Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the -galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1–99.3%), comparable with interspecific homology (94.8–100%) of S. cerevisiae MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae(MELj) was 76.2–81.7%, suggesting certain species specificity. On this evidence, the -galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.     

Role of the α1 and α2 Integrin Cytoplasmic Domains in Cell Morphology,Motility and Responsiveness to Stimulation by the Protein Kinase C Pathway

The α₁β₁ and α₂β₁ integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the α subunits. We expressed either full-length α₁ integrin subunit cDNA (XICI), full-length α₂ integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of Q₂ subunit and the cytoplasmic domain of α₁ (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of α₁ subunit and the, cytoplasmic domain of α₂ (X1C2), α₁ cDNA truncated after the GFFKR sequence (X1C0) or α₂ cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the a_x and α₂ subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the α₁ or α₂ cytoplasmic tail. Spreading was also influenced by the presence of an α subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the α₂ cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, α₂-cytoplasmic tail-dependent migration.     

Light-Induced Lipid Peroxidation in Isolated Chloroplasts and Role of α-Tocopherol

Lipid peroxidation in isolated chloroplasts illuminated by visible light and the role of α-tocopherol in chloroplasts were studied. The TBA reactants and fluorescent products derived from lipid peroxidation were formed by illumination. Peroxidation was inhibited by free radical scavengers and ¹O₂ quenchers. Hydroxy methyl octadecanoates, which were the reduced and hydrogenated products of lipid hydroperoxides, were detected. Among them, 10-and 15-hydroxy methyl octadecanoates were generated from ¹O₂ oxidation. On the other hand, lipid hydroperoxides did not accumulate in this peroxidation process. The amount of α-tocopherol in the chloroplasts decreased with lipid peroxidation, and α-tocopheryl quinone was produced. The results indicate that α-tocopherol acts as a free radical scavenger for photo-oxidation of chloroplasts.     

Chemical Structure of Mating Pheromone Peptides, αsk1 and αsk3 Pheromones,Produced by Mating Type α Cells of Saccharomyces kluyveri

Three peptides, α^sk1, α^sk2 and α^sk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.     

α-Tocopherol and lipid profiles in plasma and the expression of α-tocopherol-related molecules in the liver of Opisthorchis viverrini-infected hamsters

Opisthorchis viverrini infection induces inflammation-mediated oxidative stress and liver injury, which may alter α-tocopherol and lipid metabolism. We investigated plasma α-tocopherol and lipid profiles in hamsters infected with O. viverrini. Levels of α-tocopherol, cholesterol, and low-density lipoprotein increased in the acute phase of infection. In the chronic phase, α-tocopherol decreased, while triglyceride and very low-density lipoprotein increased. Notably, high-density lipoprotein decreased both in the acute and chronic phases. In the liver, cholesteryl oleate, triolein, and oleic acid decreased in the acute phase, and increased in the chronic phase. Such chronological changes were negatively correlated with the plasma α-tocopherol level. The expression of α-tocopherol-related molecules, ATP-binding cassette transporter A1 (ABCA1) and α-tocopherol transfer protein, increased throughout the experiment. These results suggest that O. viverrini infection profoundly affects on lipid and α-tocopherol metabolism in due course of infection.     

The Studies on the Action of Yeast Hexokinase upon the α- and β-Diasteromers of d-Glucose

The yeast hexokinase is highly specific for α-isomer of d-glucose. The relative rate of phosphorylation of β-d-glucose, catalyzed by the purified yeast hexokinase, is observed to be 60~70 (α-d-glucose=100). The average Michaelis constants of yeast hexokinase are found to be 1.8 × 10^?4 and 2.4 × 10^?4 for α-d-glucose and (β-d-glucose respectively, therefore the difference between the two constants is considered to be negligible.