Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.     

Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes

Genome-wide association (GWA) studies have identified multiple loci at which common variants modestly but reproducibly influence risk of type 2 diabetes (T2D). Established associations to common and rare variants explain only a small proportion of the heritability of T2D. As previously published analyses had limited power to identify variants with modest effects, we carried out meta-analysis of three T2D GWA scans comprising 10,128 individuals of European descent and approximately 2.2 million SNPs (directly genotyped and imputed), followed by replication testing in an independent sample with an effective sample size of up to 53,975. We detected at least six previously unknown loci with robust evidence for association, including the JAZF1 (P = 5.0 x 10(-14)), CDC123-CAMK1D (P = 1.2 x 10(-10)), TSPAN8-LGR5 (P = 1.1 x 10(-9)), THADA (P = 1.1 x 10(-9)), ADAMTS9 (P = 1.2 x 10(-8)) and NOTCH2 (P = 4.1 x 10(-8)) gene regions. Our results illustrate the value of large discovery and follow-up samples for gaining further insights into the inherited basis of T2D.     

SLC2A9 influences uric acid concentrations with pronounced sex-specific effects

Serum uric acid concentrations are correlated with gout and clinical entities such as cardiovascular disease and diabetes. In the genome-wide association study KORA (Kooperative Gesundheitsforschung in der Region Augsburg) F3 500K (n = 1,644), the most significant SNPs associated with uric acid concentrations mapped within introns 4 and 6 of SLC2A9, a gene encoding a putative hexose transporter (effects: -0.23 to -0.36 mg/dl per copy of the minor allele). We replicated these findings in three independent samples from Germany (KORA S4 and SHIP (Study of Health in Pomerania)) and Austria (SAPHIR; Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk), with P values ranging from 1.2 x 10(-8) to 1.0 x 10(-32). Analysis of whole blood RNA expression profiles from a KORA F3 500K subgroup (n = 117) showed a significant association between the SLC2A9 isoform 2 and urate concentrations. The SLC2A9 genotypes also showed significant association with self-reported gout. The proportion of the variance of serum uric acid concentrations explained by genotypes was about 1.2% in men and 6% in women, and the percentage accounted for by expression levels was 3.5% in men and 15% in women.     

Activation and inactivation of thyroid hormone by deiodinases: Local action with general consequences

The thyroid hormone plays a fundamental role in the development, growth, and metabolic homeostasis in all vertebrates by affecting the expression of different sets of genes. A group of thioredoxin fold-containing selenoproteins known as deiodinases control thyroid hormone action by activating or inactivating the precursor molecule thyroxine that is secreted by the thyroid gland. These pathways ensure regulation of the availability of the biologically active molecule T3, which occurs in a time-and tissue-specific fashion. In addition, because cells and plasma are in equilibrium and deiodination affects central thyroid hormone regulation, these local deiodinase-mediated events can also affect systemic thyroid hormone economy, such as in the case of non-thyroidal illness. Heightened interest in the field has been generated following the discovery that the deiodinases can be a component in both the Sonic hedgehog signaling pathway and the TGR-5 signaling cascade, a G-protein-coupled receptor for bile acids. These new mechanisms involved in deiodinase regulation indicate that local thyroid hormone activation and inactivation play a much broader role than previously thought. Received 29 August 2007; received after revision 11 October 2007; accepted 16 October 2007     

Zinc binding to peptide analogs of the structural zinc site in alcohol dehydrogenase: Implications for an entatic state

Zinc binding to the peptide replica and analogs to residues 93–115 of horse liver alcohol dehydrogenase (ADH) was examined by competition of the peptides and the chromophoric chelator 4-(2- pyridylazo)resorcinol for zinc and X-ray absorption fine structure analysis of the zinc ligands. In the enzyme, zinc is coordinated by four Cys residues. In the peptide replica, zinc is bound to three Cys and one His residue. A four-Cys zinc coordination is observed only when His is removed, leading to increased zinc stability. ADH crystal structures reveal that the ε-amino group of the conserved residue Lys323 is within H-bond distance of the backbone amide oxygens of residues 103, 105 and 108, likely stabilizing the zinc coordination in the enzyme. The peptide data thus indicate structural strain and increased energy in the zinc-binding site in the protein, characteristic of an entatic state, implying a functional nature for this zinc site. Received 3 July 2008; received after revision 11 August 2008; accepted 1 September 2008     

Common variants on 1p36 and 1q42 are associated with cutaneous basal cell carcinoma but not with melanoma or pigmentation traits

To search for new sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conducted a genome-wide SNP association study of 930 Icelanders with BCC and 33,117 controls. After analyzing 304,083 SNPs, we observed signals from loci at 1p36 and 1q42, and replicated these associations in additional sample sets from Iceland and Eastern Europe. Overall, the most significant signals were from rs7538876 on 1p36 (OR = 1.28, P = 4.4 x 10(-12)) and rs801114 on 1q42 (OR = 1.28, P = 5.9 x 10(-12)). The 1p36 locus contains the candidate genes PADI4, PADI6, RCC2 and ARHGEF10L, and the gene nearest to the 1q42 locus is the ras-homolog RHOU. Neither locus was associated with fair pigmentation traits that are known risk factors for BCC, and no risk was observed for melanoma. Approximately 1.6% of individuals of European ancestry are homozygous for both variants, and their estimated risk of BCC is 2.68 times that of noncarriers.     

A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse

In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gray horses. The Gray horse provides a notable example of how humans have cherry-picked mutations with favorable phenotypic effects in domestic animals.