Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na+/Pi Cotransporter

The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants.     

Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1alphaOHase-deficient mice

Intake of a low-phosphate diet stimulates transepithelial transport of P_i in small intestine as well as in renal proximal tubules. In both organs, this is paralleled by a change in the abundance of the apically localized NaP_i cotransporters NaP_i type IIa (NaP_i-IIa) and NaP_i type IIb (NaP_i-IIb), respectively. Low-P_i diet, via stimulation of the activity of the renal 25-hydroxyvitamin-D₃-1-hydroxylase (1OHase), leads to an increase in the level of 1,25-dihydroxy-vitamin D₃ [1,25(OH)₂D]. Regulation of the intestinal absorption of P_i and the abundance of NaP_i-IIb by 1,25(OH)₂D has been supposed to involve the vitamin D receptor (VDR). In this study, we investigated the adaptation to a low-P_i diet of NaP_i-IIb in small intestine as well as NaP_i-IIa in kidneys of either VDR- or 1OHase-deficient mice. In both mouse models, upregulation by a low-P_i diet of the NaP_i cotransporters NaP_i-IIa and NaP_i-IIb was normal, i.e., similar to that observed in the wild types. Also, in small intestines of VDR- and 1OHase-deficient mice, the same changes in NaP_i-IIb mRNA found in wild-type mice were observed. On the basis of the results, we conclude that the regulation of NaP_i cotransport in small intestine (via NaP_i-IIb) and kidney (via NaP_i-IIa) by low dietary intake of P_i cannot be explained by the 1,25(OH)₂D-VDR axis. NaP_i type IIb; vitamin D₃     

A conserved N-capping motif contributes significantly to the stabilization and dynamics of the C-terminal region of class Alpha glutathione S-transferases

Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state.     

Acute regulation of Na/H exchanger NHE3 by adenosine A(1) receptors is mediated by calcineurin homologous protein

Adenosine is an autacoid that regulates renal Na(+) transport. Activation of adenosine A(1) receptor (A(1)R) by N(6)-cyclopentidyladenosine (CPA) inhibits the Na(+)/H(+) exchanger 3 (NHE3) via phospholipase C/Ca(2+)/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A(1)R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10(-6) M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3(C terminus); prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3(C terminus) (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3(delta552)) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3(462-552)) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3(Full-length) interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A(1)R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.     

Relations of APOE promoter polymorphisms to LDL cholesterol and markers of subclinical atherosclerosis in young adults

The common apolipoprotein E (apoE) gene (APOE) epsilon2/epsilon3/epsilon4 polymorphism explains part of serum lipid variation, and polymorphisms in the APOE promoter region have been proposed to participate in the regulation of serum lipid levels within the most common APOE epsilon3/epsilon3 genotype group. We determined APOE -219G/T and +113G/C promoter genotypes and estimated APOE haplotypes in 525 participants of the Cardiovascular Risk in Young Finns Study. We studied the associations of the APOE promoter polymorphisms and their haplotypes with cross-sectional and longitudinal serum lipid and apolipoprotein concentrations as well as with flow-mediated dilatation (FMD), carotid artery compliance (CAC), and intima-media thickness (IMT) within the APOE epsilon3/epsilon3 carriers. We found no significant association between the APOE promoter genotypes and serum lipids [low density lipoprotein-cholesterol (LDL-C), HDL-C, and triglycerides], apolipoproteins (apoA-I and apoB), or brachial artery FMD, CAC, or carotid IMT in either men or women. In longitudinal analyses in males, the carriers of heterozygous genotypes (-219G/T or +113G/C) and, furthermore, carriers of the -219T/+113C/epsilon3 haplotype had significantly higher LDL-C and total cholesterol concentrations throughout the 21 year follow-up period compared with homozygous G allele carriers or noncarriers of the -219T/+113C/epsilon3 haplotype. Such associations were not found in females. In summary, the APOE promoter polymorphisms -219G/T and +113G/C as well as their haplotype are associated with longitudinal changes in LDL-C and total cholesterol concentrations in young Finnish males but do not seem to be major determinants for FMD, CAC, or carotid IMT in males or females.     

Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating

Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins'' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.     

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.