An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.     

A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels

Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca2+-binding proteins termed KChIPs with pore-forming Kv4 alpha subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). Here we show that KChIPs profoundly affect the intracellular trafficking and molecular properties of Kv4.2 alpha subunits. Co-expression of KChIPs1-3 causes a dramatic redistribution of Kv4.2, releasing intrinsic endoplasmic reticulum retention and allowing for trafficking to the cell surface. KChIP co-expression also causes fundamental changes in Kv4.2 steady-state expression levels, phosphorylation, detergent solubility, and stability that reconstitute the molecular properties of Kv4.2 in native cells. Interestingly, the KChIP4a isoform, which exhibits unique effects on Kv4 channel gating, does not exert these effects on Kv4.2 and negatively influences the impact of other KChIPs. We provide evidence that these KChIP effects occur through the masking of an N-terminal Kv4.2 hydrophobic domain. These studies point to an essential role for KChIPs in determining both the biophysical and molecular characteristics of Kv4 channels and provide a molecular basis for the dramatic phenotype of KChIP knockout mice.     

Differential degradation of amyloid beta genetic variants associated with hereditary dementia or stroke by insulin-degrading enzyme

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.     

Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

The mitochondrial ornithine transporter. Bacterial expression,reconstitution, functional characterization,and tissue distribution of two human isoforms

Two isoforms of the human ornithine carrier, ORC1 and ORC2, have been identified by overexpression of the proteins in bacteria and by study of the transport properties of the purified proteins reconstituted into liposomes. Both transport L-isomers of ornithine, lysine, arginine, and citrulline by exchange and by unidirectional mechanisms, and they are inactivated by the same inhibitors. ORC2 has a broader specificity than ORC1, and L- and D-histidine, L-homoarginine, and D-isomers of ornithine, lysine, and ornithine are all substrates. Both proteins are expressed in a wide range of human tissues, but ORC1 is the predominant form. The highest levels of expression of both isoforms are in the liver. Five mutant forms of ORC1 associated with the human disease hyperornithinemia-hyperammonemia-homocitrullinuria were also made. The mutations abolish the transport properties of the protein. In patients with hyperornithinemia-hyperammonemia-homocitrullinuria, isoform ORC2 is unmodified, and its presence compensates partially for defective ORC1.     

Molecular interaction and enzymatic activity of macrophage migration inhibitory factor with immunorelevant peptides

Disulfide reduction is an important step in antigen processing for HLA class II restricted T cell responses. Migration inhibitory factor (MIF) is a member of the thioredoxin family and has been classically defined as a cytokine. Using enzyme-linked immunosorbent assay and CD analysis, here we describe the binding to MIF of two peptides, hepatitis B surface antigen (HBsAg) and insulin B (InsB) with high affinity for HLA class II allo-types, HLA-DP2 and HLA-DQ8, respectively. At neutral pH, cysteinylated InsB was a substrate for MIF thiol reductase activity, as assessed by mass spectroscopy/electrospray analysis. Finally, a biologically active form of MIF co-immunopurified with mature forms of HLA DP2/15, and a peptide derived from the HLA-DP beta1 helix could be used for affinity purification of MIF. The possibility that MIF participates in class II antigen presentation and/or as a chaperone is discussed.     

BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.     

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver,kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.     

Myotonic dystrophy type 2: human founder haplotype and evolutionary conservation of the repeat tract

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.     

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.