Functional consequences of single nucleotide polymorphisms in the human organic anion transporter hOAT1 (SLC22A6).

The human organic anion transporter hOAT1 (SLC22A6) contributes to the uptake of a range of small organic anions across the basolateral membrane of the renal proximal tubule and drives their urinary elimination. The aim of this study was to identify genetic variants of hOAT1 and to investigate potential effects on the functional properties of this transporter. Twenty single nucleotide polymorphisms (SNPs) in hOAT1 were identified in genomic DNA from 92 individuals of African, Asian, and Caucasian origin. Two SNPs encoded changes in amino acid sequence; arginine to histidine (residue 50) and lysine to isoleucine (residue 525). Significantly, these SNPs were only present in the samples of African origin. When expressed in Xenopus oocytes, wild-type R50-hOAT1 and the variants R50H-hOAT1 and K525I-hOAT1 all mediated the probenecid-sensitive uptake of the classic organic anion para-aminohippurate (PAH). Kinetic analysis indicated that the transport affinity (K(m)) for PAH was unchanged in the variants, compared with wild type. Interestingly, the K(m) for the nucleoside phosphonate analogs adefovir, cidofovir, and tenofovir seemed to be decreased in the R50H-hOAT1 variant compared with the wild type, whereas the kinetics of K525I-hOAT1 remained unchanged. In conclusion, this is the first study to identify variation of hOAT1 in a racially diverse sample and to investigate the functional properties of the resulting variants. Since hOAT1 has been suggested as the basis of nephrotoxicity induced by nucleoside phosphonate analogs, this study raises the intriguing possibility that individuals with genetic variation in hOAT1, such as R50H, may display different handling of these drugs.

Selecton: a server for detecting evolutionary forces at a single amino-acid site.

UNLABELLED: We present an algorithmic tool for the identification of biologically significant amino acids in proteins of known three dimensional structure. We estimate the degree of purifying selection and positive Darwinian selection at each site and project these estimates onto the molecular surface of the protein. Thus, patches of functional residues (undergoing either positive or purifying selection), which may be discontinuous in the linear sequence, are revealed. We test for the statistical significance of the site-specific scores in order to obtain reliable and valid estimates. AVAILABILITY: The Selecton web server is available at: http://selecton.bioinfo.tau.ac.il SUPPLEMENTARY INFORMATION: More information is available at http://selecton.bioinfo.tau.ac.il/overview.html. A set of examples is available at http://selecton.bioinfo.tau.ac.il/gallery.html.

Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites.

Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted. Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants). The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed. Among 14 mutants, three beta-2 mutants (Y26A, Y79A, and Y181A), and three beta-3 mutants (Y92A, W149A, and Y241A) showed decreased activity. These mutated residues resided at Sites 1, 2, and 4, at the same locations relatively in the binding sites. Mutagenesis of Tyr or Trp at the corresponding locations in Sites 3 and 5 did not lead to a reduction in activity. Results indicate that the properties of Sites 1, 2, and 4 are different from those of Sites 3 and 5, and that the contribution of these two sites to the hemagglutination reaction was minor.

Repression of the steroidogenic acute regulatory gene by the multifunctional transcription factor Yin Yang 1.

The transport of cholesterol to the inner mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein is a critical step in steroidogenesis. The current study was designed to examine whether the multifunctional transcription factor Yin Yang 1 (YY1) was able to repress activation of the StAR gene. YY1 bound to three putative YY1-binding sites in the rat StAR promoter. Cotransfection of the StAR promoter linked to a luciferase reporter gene and YY1 in the presence or absence of sterol regulatory element binding protein-1a (SREBP-1a) resulted in transcriptional repression. YY1 was found to colocalize in the nucleus with SREPB-1a. YY1 inhibited SREBP-1a/nuclear factor Y (NF-Y) enhancement of StAR activation and YY1 decreased SREBP-1a binding to a sterol regulatory element in the presence or absence of NF-Y. YY1 also decreased NF-Y binding to a nonconsensus NF-Y-binding motif in the StAR promoter. These studies provide novel information on the mechanisms of YY1-mediated repression of the StAR gene.

The ORFO product of Potato leafroll virus is indispensable for virus accumulation.

Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.

Amino acid substitution within the S2 and S4 transmembrane segments in Shaker potassium channel modulates channel gating.

To investigate of the gating properties in the voltage-activated potassium channel, we have mutated a variety of S2 and S4 residues in the Shaker potassium protein. Results showed that the R365C and R368C, but not the E283C, R362C, R365S, R368S or the ShB-IR, were sensitive to micromolar concentrations of Cd(2+) ions. This indicates that R365 and R368 play a crucial role in the channel gating due to a conformational modulation of the channel structure. Doubly mutated channels of the E283C/R365E and E283C/R368E caused a transient increase in current amplitude, which reached a peak within a few seconds and then decreased toward initial levels, despite the continual presence of Cd(2+). Taken together, our results suggest that E283, R365, and R368 form a network of strong, local, and electrostatic interactions that relate closely to the mechanism of the channel gating.

Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

A parallel binding motif 16mer triplex-forming oligonucleotide (TFO) complementary to a polypurine-polypyrimidine target region near the 3'-end of the SupF gene of plasmid pSP189 was labeled with [5-(125)I]dCMP at position 15. Following triplex formation and decay accumulation, radiation-induced site-specific double-strand breaks (DSBs) were produced in the pSP189 SupF gene. Bulk damaged DNA and the isolated site-specific DSB-containing DNA were separately transfected into human WI38VA13 cells and allowed to repair prior to recovery and analysis of mutants. Bulk damaged DNA had a relatively low mutation frequency of 2.7 x 10(-3). In contrast, the isolated linear DNA containing site-specific DSBs had an unusually high mutation frequency of 7.9 x 10(-1). This was nearly 300-fold greater than that observed for the bulk damaged DNA mixture, and >1.5 x 10(4)-fold greater than background. The mutation spectra displayed a high proportion of deletion mutants targeted to the(125)I binding position within the SupF gene for both bulk damaged DNA and isolated linear DNA. Both spectra were characterized by complex mutations with mixtures of changes. However, mutations recovered from the linear site-specific DSB-containing DNA presented a much higher proportion of complex deletion mutations.

Subunit folding and assembly steps are interspersed during Shaker potassium channel biogenesis.

In the voltage-dependent Shaker K+ channel, distinct regions of the protein form the voltage sensor, contribute to the permeation pathway, and recognize compatible subunits for assembly. To investigate channel biogenesis, we disrupted the formation of these discrete functional domains with mutations, including an amino-terminal deletion, Delta97-196, which is likely to disrupt subunit oligomerization; D316K and K374E, which prevent proper folding of the voltage sensor; and E418K and C462K, which are likely to disrupt pore formation. We determined whether these mutant subunits undergo three previously identified assembly events as follows: 1) tetramerization of Shaker subunits, 2) assembly of Shaker (alpha) and cytoplasmic beta subunits, and 3) association of the amino and carboxyl termini of adjacent Shaker subunits. Delta97-196 subunits failed to establish any of these quaternary interactions. The Delta97-196 deletion also prevented formation of the pore. The other mutant subunits assembled into tetramers and associated with the beta subunit but did not establish proximity between the amino and carboxyl termini of adjacent subunits. The results indicate that oligomerization mediated by the amino terminus is required for subsequent pore formation and either precedes or is independent of folding of the voltage sensor. In contrast, the amino and carboxyl termini of adjacent subunits associate late during biogenesis. Because subunits with folding defects oligomerize, we conclude that Shaker channels need not assemble from pre-folded monomers. Furthermore, association with native subunits can weakly promote the proper folding of some mutant subunits, suggesting that steps of folding and assembly alternate during channel biogenesis.

Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135.

The role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site-directed mutagenesis, hemagglutination-inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl alpha-N-dansylgalactosaminide (MealphaGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL-MealphaGalNDns complex. Docking of MealphaGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MealphaGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MealphaGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed in high-temperature dynamics simulations, appears not to be a required step.

Mutagenesis of rat dopamine beta-hydroxylase: examination in cell-free system.

Dopamine beta-hydroxylase (DBH; EC 1.14.17.1) exists as membrane-bound and soluble forms in neurosecretory vesicles. The features of DBH required for glycosylation and incorporation into membranes were studied in a cell-free system. Translation of full-length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl-terminal, in contrast to amino-terminal, truncations gave translation products that were glycosylated by microsomal membranes. Site-directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavage site modified. Translation without membranes indicated that the second AUG is not used to initiate translation. The mutant with Glu41-->Leu41 and Ser43-->Thr43 yielded only the GH form with membranes, whereas mutation of Ser43-->Ala43 generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membranes on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moiety. The results suggest a role for cleavage of a signal sequence in the formation of different forms of DBH. The possibility that these mutations change the secondary structure near the signal cleavage site, affecting processing, is discussed.

Transfection with a cDNA encoding a Ser31 or Ser34 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance.

Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.

Copper-dependent site-specific mutagenesis by benzoyl peroxide in the supF gene of the mutation reporter plasmid pS189.

Benzoyl peroxide (BzPO) enhances tumor promotion and malignant conversion in mouse epidermis. DNA damage may contribute to these processes. BzPO reacts with Cu(I) to produce the benzoyloxyl radical, which in turn causes strand breaks in plasmid DNA. In this study we investigated whether BzPO with or without Cu(I) caused promutagenic DNA damage in the supF gene of the mutation reporter plasmid pS189 replicating in human Ad293 cells. Exposure of pS189 in vitro to BzPO (0.1-1 mM) inhibited plasmid replication; however, addition of Cu(I) (0.1 mM) did not augment BzPO-induced plasmid toxicity. Exposure to BzPO with or without 0.1 mM Cu(I) was also associated with a concentration-dependent increase in mutation frequency, up to > 100-fold above the spontaneous mutation frequency. Supplemental Cu(I) was not required for mutagenesis; however, it both raised the maximal mutation frequency observed and lowered the threshold concentration of BzPO necessary to discern mutagenesis above the spontaneous background. Neither the hydroxyl radical scavengers mannitol or DMSO, the spin trap N-tert-butyl-alpha-phenylnitrone, nor reduced glutathione altered BzPO/Cu(I)-induced mutagenesis; however, mutagenesis was suppressed by the chelator EDTA. Twenty-four of 32 individual BzPO/Cu(I)-induced mutants characterized by sequencing contained point mutations; 22/25 point mutations occurred at G-C base pairs. There were five large deletions and four small deletions. Three additional BzPO-induced mutants contained four point mutations, all occurring at G-C base pairs. Two BzPO/Cu(I)-induced mutational clusters at d(pGGG)-d(pCCC) sites were observed. These data suggest that BzPO may interact with Cu(I) bound to G-C base pairs in DNA to produce site-specific promutagenic DNA damage.