Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization.

Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin alpha contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin alpha also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

Comparative aspects of the function and mechanism of SUR1 and MDR1 proteins.

ATP-binding cassette (ABC) superfamily proteins have divergent functions and can be classified as transporters, channels, and receptors, although their predicted secondary structures are very much alike. Prominent members include the sulfonylurea receptor (SUR1) and the multidrug transporter (MDR1). SUR1 is a subunit of the pancreatic beta-cell K(ATP) channel and plays a key role in the regulation of glucose-induced insulin secretion. SUR1 binds ATP at NBF1, and ADP at NBF2 and the two NBFs work cooperatively. The pore-forming subunit of the pancreatic beta-cell K(ATP) channel, Kir6.2, is a member of the inwardly rectifying K(+) channel family, and also binds ATP. In this article, we present a model in which the activity of the K(ATP) channel is determined by the balance of the action of ADP, which activates the channel through SUR1, and the action of ATP, which stabilizes the long closed state by binding to Kir6.2. The concentration of ATP could also affect the channel activity through binding to NBF1 of SUR1. MDR1, on the other hand, is an ATP-dependent efflux pump which extrudes cytotoxic drugs from cells before they can reach their intracellular targets, and in this way confers multidrug resistance to cancer cells. Both NBFs of MDR1 can hydrolyze nucleotides, and their ATPase activity is necessary for drug transport. The interaction of SUR1 with nucleotides is quite different from that of MDR1. Variations in the interactions with nucleotides of ABC proteins may account for the differences in their functions.

Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy: nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1.

To elucidate the genetic etiology of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) in the Japanese population, we conducted a polymerase chain reaction-single-strand conformation polymorphism analysis of the sulfonylurea receptor 1 (SUR1) and Kir6.2 genes in 17 Japanese PHHI patients, including a pair of siblings from a consanguineous family. We also analyzed the glutamate dehydrogenase gene for the exons encoding an allosteric regulatory domain of the enzyme. In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. None of these mutations were found in control Japanese subjects. Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Functional consequences of these mutations on K(ATP) function were evaluated using 86Rb+ efflux studies in COS-7 cells. SUR1-446fsdelT and SUR1-1436Q did not form a functional K(ATP). Western blot analysis after transient expression in COS-7 cells revealed the expression of SUR1-1436Q protein to be markedly reduced, suggesting SUR1-1436Q to be unstable in these cells. K(ATP)(SUR1-1420C) showed reduced responses to metabolic inhibition by oligomycin and 2-deoxyglucose. K(ATP) channels are under complex regulation by intracellular ATP and ADP. ATP both inhibits and activates these channels. The inhibition is probably mediated through direct ATP interaction with a pore-forming subunit Kir6.2, whereas the activation is likely to be through a regulatory subunit SUR1. There is a cooperative regulation of ATP and ADP binding to SUR1, and this cooperativity may be involved in regulating the K(ATP) channel. In SUR1-1420C, high-affinity binding of ATP to the nucleotide-binding fold (NBF)-1 was indistinguishable from that of wild-type SUR1. However, stabilization of ATP binding to NBF-1 by MgATP or MgADP was impaired, suggesting that this defect may account for impaired K(ATP)(SUR1-1420C) function. This is the first direct biochemical evidence that the cooperativity of nucleotide binding to SUR1 is impaired in a SUR1 mutant causing PHHI. No mutations were identified in the Kir6.2 and glutamate dehydrogenase genes. The genetic etiology of PHHI appears to be heterogeneous. SUR1 mutations may account for no more than 20% of PHHI cases in Japanese patients. Mutations of Kir6.2 and glutamate dehydrogenase genes are likely to be even less common.

Crystal structure of the fungal peroxidase from Arthromyces ramosus at 1.9 A resolution. Structural comparisons with the lignin and cytochrome c peroxidases.

The crystal structure of the peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) from the hyphomycete Arthromyces ramosus (ARP) has been determined by the multiple isomorphous replacement method and refined by the simulated annealing method to a crystallographic R-factor of 17.4% for the 19,191 reflections with F > 2 sigma F between 7.0 and 1.9 A resolution. The model includes residues 9 to 344, the heme group, two N-acetylglucosamine residues, two calcium ions and 246 water molecules. The root-mean-square deviation of bond lengths from the ideal values is 0.02 A. The mean coordinate error is estimated as 0.2 A. The electron density of the glycine-rich region of the amino-terminal eight residues was invisible. ARP has ten major and two short alpha-helices and a few short beta-strands. The overall tertiary structure of ARP is similar to that of yeast cytochrome c peroxidase (CCP) and is particularly similar to that of the lignin peroxidase (LiP) from Phanerochaete chrysosporium. Relative to CCP, ARP and LiP each have an extension of approximately 40 residues at the carboxy terminus. All eight cysteine residues in ARP form disulfide bonds (C12:C24, C23:C293, C43:C129 and C257:C322). Two calcium sites are inaccessible to solvent. The four disulfide bonds and two calcium sites, which are lacking in CCP, are conserved in ARP and LiP. The bond from Asn304C to Ala305N in ARP is the site sensitive to proteases. An Asx turn present in the Asn303 to Ala305 segment appears to orient the side-chain of Asn304 to outward from the molecule, rendering it easily trappable by pockets of proteases. The proximal heme ligand is His184 in helix F (distance of N epsilon 2 ... Fe, 2.10 A), and one of several water molecules in the distal pocket of the heme bridges the iron atom and the N epsilon 2 of His56. The orientation of the imidazole ring of the distal histidine residue relative to the heme group in ARP differs significantly from that in LiP. The access channel to the distal side of the heme of ARP is markedly wider along the heme plane than that of LiP. Many of the amino acid residues that comprise the entrance of this channel differ for ARP and LiP. This may account for the differences in substrate specificity.

Expression of vinexin alpha in the dorsal half of the eye and in the cardiac outflow tract and atrioventricular canal.

Vinexin, a recently identified cytoskeletal protein, contains three SH3 domains and plays important roles in regulation of cytoskeletal organization and signal transduction. Using whole-mount in situ hybridization, we showed here that expression of vinexin alpha, the longer vinexin transcript, is strictly regulated, although the shorter transcript, vinexin beta, is expressed almost ubiquitously during embryonic development in mice. Expression of vinexin alpha was limited to within part of the eye and heart in 10.5 dpc embryos. Analysis of cryosections of 10.5 dpc embryos showed that vinexin alpha was expressed in a dorsal half of the retinal pigment epithelium and in the outflow tract and atrioventricular canal of the heart. Furthermore, we also found that vinexin alpha was expressed in the gonad and in a ventral part of the pons of 12.5 dpc embryos. These results indicated that the expression of vinexin alpha is strictly regulated in a temporally and spatially restricted manner.

CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line.

A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth.

NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1.

Pancreatic beta-cell ATP-sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regulate glucose-induced insulin secretion. To learn more about the interaction of SUR1 with nucleotides, we examined the effect of N-ethylmaleimide (NEM) modification. Photoaffinity labeling of SUR1 with 5 microM 8-azido-[alpha-32P]ATP or 8-azido-[gamma-32P]ATP was inhibited by NEM with Ki of 1.8 microM and 2.4 microM, and Hill coefficients of 0.94 and 1.1, respectively. However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 microM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1.

Inhibition of oxygen toxicity by targeting superoxide dismutase to endothelial cell surface.

Since enzymes that degrade reactive oxygens, such as superoxide dismutase (SOD), are significantly lower in plasma than in intracellular compartments, cell surface membranes should be protected against hazardous oxygens particularly when animals are challenged with oxidative stress. To minimize oxygen toxicity on endothelial cell surface, a fusion gene consisting of cDNA coding human Cu2+/Zn2(+)-SOD and heparin-binding peptide was constructed and expressed in yeast. The resulting enzyme (HB-SOD) bound to a heparin-Sepharose column and cultured endothelial cells; binding was inhibited either by high NaCl concentrations or heparin. When injected intravenously, HB-SOD predominantly bound to vascular endothelial cell surface. Carrageenin-induced paw edema and cold-induced brain edema of the rat were markedly inhibited by a single dose of HB-SOD. These results suggest that superoxide radical and/or its metabolite(s) occurring at or near the outer surface of vascular endothelial cells might play a critical role in the pathogenesis of vasogenic edema.

Site-directedly mutated human cytochrome c which retains heme c via only one thioether bond.

Although Cys-14 (human numbering) of cytochrome c was conserved during its molecular evolution and it is supposed to be essential for most cytochromes c to retain heme c via two thioether bonds, a site-directedly mutated human cytochrome c which has an alanine residue at this position and only one thioether bond through Cys-17 turns out to be functional. This shows that Cys-14 is not essential. The absorption spectrum of the atypical cytochrome c is red shifted, and similar to those of Euglena and Crithidia cytochromes c, which also have only one thioether bond [Pettigrew, G.W., Leaver, J.L., Meyer, T.E., & Ryle, A.P. (1975) Biochem. J. 147, 291-302].

Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae.

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.

Amino acid replacement studies of human cytochrome c by a complementation system using CYC1 deficient yeast.

Various in vitro mutated human cytochrome c genes which encode displaced amino acid residues at the 14th, 17th, 28th, 37th, 38th, 56th, and/or 84th residues were constructed, and their degrees of complementation of yeast CYC1 deficiency were examined. Invariant Cys-17 and Arg-38 could not be replaced by alanine and tryptophan, respectively, without function impairment. Cytochrome c containing Ala-14 instead of conserved Cys-14, Gly-38 or Lys-38 instead of Arg-38, and Ser-84 instead of invariant Gly-84 were partly functional. These results indicate that these invariant or conserved residues are important. Cytochromes c containing Cys-56 instead of native Gly-56 was partly functional. Cytochrome c containing Arg-37 and Gly-38 instead of Gly-37 and Arg-38 was slightly functional. Replacement of variable Thr-28 and Gly-37 by Ile-28 and Arg-37, respectively, produced no effects. Our results are as a whole consistent with the view that conserved residues are important and variable residues are less important for cytochrome c to function.

Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate.

The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.

Purification, characterization, and application of an acid urease from Arthrobacter mobilis.

It has been shown that urea in fermented beverages and foods can serve as a precursor of ethylcarbamate, a potential carcinogen, and acid urease is an effective agent for removing urea in such products. We describe herein the purification and characterization of a novel acid urease from Arthrobacter mobilis SAM 0752 and show its unique application for the removal of urea from fermented beverages using the Japanese rice wine, sake, as an example. The purified acid urease showed an optimum pH for activity at pH 4.2. The enzyme exhibited an apparent K(m) for urea of 3.0 mM and a Vmax of 2370 mumol of urea per mg and min at 37 degrees C and pH 4.2. Gel permeation chromatographic and sodium dodecyl sulfate gel electrophoretic analyses showed that the enzyme has an apparent native molecular weight (M(r)) of 290,000 and consisted of three types of subunit proteins (M(r), 67,000, 16,600, 14,100) denoted by alpha, beta, and gamma. The most probable stoichiometry of the subunits was estimated to be alpha: beta: gamma = 1:1:1, suggesting the enzyme subunit structure of (alpha beta gamma)3. The enzyme also existed as an aggregated form with an M(r) of 580,000. The purified enzyme contained 2 g-atom of nickel per alpha beta gamma unit of the enzyme. Enzyme activity was inhibited by acetohydroxamic acid, HgCl2, and CuCl2. The isoelectric point of the native enzyme was estimated by gel electrofocusing to be 6.8. Urea (50 ppm), which was exogenously added to sake (pH 4.4, 17 +/- 1% (v/v) ethanol), was completely decomposed by incubation with the enzyme (0.09 U ml-1) at 15 degrees C for 13 days. The enzyme was unstable at temperatures higher than 65 degrees C and pHs lower than 4, and was completely inactivated under the conditions of a pasteurization step involved in the traditional sake-making processes. These results indicate that the enzyme is applicable to the elimination of urea in fermented beverages with minimal modification to the conventional process.

Comparative studies of suidatrestin, a specific inhibitor of trehalases.

Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor. Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases. The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast. Suidatrestin was inactive on maltase but inhibited all trehalases with IC50 values of 0.08-0.1 microM; Ki values ranged from 0.02 to 0.05 microM. The very low Ki/K(m) ratios (3.9 x 10(-6) -4.9 x 10(-6)) indicated excellent in vitro inhibitory action of suidatrestin. When injected into larvae of S. littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only. Consecutive omission of the inhibitor even stimulated weight increase above that of controls. Significant mortality was achieved at a rather high dose only. Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration. The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.

Effects of oleic, arachidonic and 5,8,11,14-nonadecatetraenoic acids on lipid secretion and ketogenesis in perfused rat liver.

The effects of perfused oleic (18:1n-9), arachidonic (20:4n-6) and 5,8,11,14-nonadecatetraenoic (19:4n-5) acids on triglyceride and cholesterol secretion and ketone body production were studied in isolated rat liver. As compared to oleic and 19:4n-5 acids, both ketone body production and triglyceride secretion were significantly lowered when arachidonic acid was perfused. The concentration of triglyceride in the post-perfused liver was lower upon perfusion with arachidonic acid than upon perfusion with oleic acid or 19:4n-5 acid. Cholesterol secretion in the liver perfused with arachidonic acid or 19:4n-5 acid was significantly higher than with oleic acid. The concentration of cholesterol in the post-perfused liver was slightly but significantly higher with 19:4n-5 acid than with the other fatty acids. The results suggest that 19:4n-5 acid when compared with arachidonic acid affects lipid metabolism in liver differently.

HPLC analysis and optimization of enzymatic synthesis of 4'-O-(beta-D-glucopyranosyl)-D-pantothenic acid.

We analyzed beta-glucosidase-catalyzed transglucosylation to D-pantothenic acid using a reversed-phase HPLC system in order to obtain 4'-O-(beta-D-glucopyranosyl)-D-pantothenic acid (PaG) at a higher yield. The HPLC system was simpler and more straight-forward for the PaG analysis than the previously employed bioassay method and could also be adopted for efficient isolation of PaG. Penicillium decumbens naringinase showed the highest glucosyl transfer activity to D-pantothenic acid, and the reaction using smaller amounts of naringinase for prolonged periods of reaction time (70 h<) was important to attain higher yields of glucosyl transfer. Maximum overall yields of PaG of 10 and 4% (mol/mol, based on D-pantothenic acid) were obtained using beta, beta'-trehalose and cellobiose, respectively, as glucosyl donors. The value was 3.6- and 1.4-times higher, respectively, than that obtained by previous synthesis and isolation procedures.

Recent progress in P-glycoprotein research.

P-glycoprotein can extrude a variety of structurally diverse, toxic xenobiotic compounds from cells. It is believed to be one of key molecules which can cause multidrug resistance in cancer. This paper deals with recent progress in P-glycoprotein research, especially in its structure, mechanisms for substrate recognition and transport. The review also discusses specific modulators of multidrug resistance in cancer and gene therapy using the MDR1 gene.