Folding mechanism of an ankyrin repeat protein: scaffold and active site formation of human CDK inhibitor p19(INK4d)

The p19(INK4d) protein consists of five ankyrin repeats (ANK) and controls the human cell cycle by inhibiting the cyclin D-dependent kinases (CDK) 4 and 6. We investigated the folding of p19(INK4d) by urea-induced unfolding transitions, kinetic analyses of unfolding and refolding, including double-mixing experiments and a special assay for folding intermediates. Folding is a sequential two-step reaction via a hyperfluorescent on-pathway intermediate. This intermediate is present under all conditions, during unfolding, refolding and at equilibrium. The folding mechanism was confirmed by a quantitative global fit of a consistent set of equilibrium and kinetic data revealing the thermodynamics and intrinsic folding rates of the different states. Surprisingly, the N<-->I transition is much faster compared to the I<-->U transition. The urea-dependence of the intrinsic folding rates causes population of the intermediate at equilibrium close to the transition midpoint. NMR detected hydrogen/deuterium exchange and the analysis of truncated variants showed that the C-terminal repeats ANK3-5 are already folded in the on-pathway intermediate, whereas the N-terminal repeats 1 and 2 are not folded. We suggest that during refolding, repeats ANK3-ANK5 first form the scaffold for the subsequent assembly of repeats ANK1 and ANK2. The binding function of p19(INK4d) resides in the latter repeats. We propose that the graded stability and the facile unfolding of repeats 1 and 2 is a prerequisite for the down-regulation of the inhibitory activity of p19(INK4d) during the cell-cycle.     

Bipolar disorder risk alleles in adult ADHD patients

Attention-deficit/hyperactivity disorder (ADHD) has an estimated prevalence of 3-5% in adults. Genome-wide association (GWA) studies have not been performed in adults with ADHD and studies in children have so far been inconclusive, possibly because of the small sample sizes. Larger GWA studies have been performed on bipolar disorder (BD) and BD symptoms, and several potential risk genes have been reported. ADHD and BD share many clinical features and comorbidity between these two disorders is common. We therefore wanted to examine whether the reported BD genetic variants in CACNA1C, ANK3, MYO5B, TSPAN8 and ZNF804A loci are associated with ADHD or with scores on the Mood Disorder Questionnaire (MDQ), a commonly used screening instrument for bipolar spectrum disorders. We studied 561 adult Norwegian ADHD patients and 711 controls from the general population. No significant associations or trends were found between any of the single nucleotide polymorphisms (SNPs) studied and ADHD [odds ratios (ORs) ≤ 1.05]. However, a weak association was found between rs1344706 in ZNF804A (OR = 1.25; P = 0.05) and MDQ. In conclusion, it seems unlikely that these six SNPs with strong evidence of association in BD GWA studies are shared risk variants between ADHD and BD.     

Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification

The catabolism of ATP and other nucleotides participates partly in the important function of nucleotide salvage by activated cells and also in removal or de novo generation of compounds including ATP, ADP, and adenosine that stimulate purinergic signaling. Seven nucleotide pyrophosphatase/phosphodiesterase NPP family members have been identified to date. These isoenzymes, related by up conservation of catalytic domains and certain other modular domains, exert generally non-redundant functions via distinctions in substrates and/or cellular localization. But they share the capacity to hydrolyze phosphodiester or pyrophosphate bonds, though generally acting on distinct substrates that include nucleoside triphosphates, lysophospholipids and choline phosphate esters. PP_i generation from nucleoside triphosphates, catalyzed by NPP1 in tissues including cartilage, bone, and artery media smooth muscle cells, supports normal tissue extracellular PP_i levels. Balance in PP_i generation relative to PP_i degradation by pyrophosphatases holds extracellular PP_i levels in check. Moreover, physiologic levels of extracellular PP_i suppress hydroxyapatite crystal growth, but concurrently providing a reservoir for generation of pro-mineralizing P_i. Extracellular PP_i levels must be supported by cells in mineralization-competent tissues to prevent pathologic calcification. This support mechanism becomes dysregulated in aging cartilage, where extracellular PP_i excess, mediated in part by upregulated NPP1 expression stimulates calcification. PP_i generated by NPP1modulates not only hydroxyapatite crystal growth but also chondrogenesis and expression of the mineralization regulator osteopontin. This review pays particular attention to the role of NPP1-catalyzed PP_i generation in the pathogenesis of certain disorders associated with pathologic calcification.     

A de novo interstitial deletion of 8p11.2 including ANK1 identified in a patient with spherocytosis, psychomotor developmental delay, and distinctive facial features

The contiguous gene syndrome involving 8p11.2 is recognized as a combined phenotype of both Kallmann syndrome and hereditary spherocytosis, because the genes responsible for these 2 clinical entities, the fibroblast growth factor receptor 1 (FGFR1) and ankyrin 1 (ANK1) genes, respectively, are located in this region within a distance of 3.2Mb. We identified a 3.7Mb deletion of 8p11.2 in a 19-month-old female patient with hereditary spherocytosis. The identified deletion included ANK1, but not FGFR1, which is consistent with the absence of any phenotype or laboratory findings of Kallmann syndrome. Compared with the previous studies, the deletion identified in this study was located on the proximal end of 8p, indicating a pure interstitial deletion of 8p11.21. This patient exhibited mild developmental delay and distinctive facial findings in addition to hereditary spherocytosis. Thus, some of the genes included in the deleted region would be related to these symptoms.     

酵母PHO81蛋白的结构预测和功能分析

以Ｃｈｏｕ－Ｆａｓｍａｎ和ＧＯＲ方法预测酵母ＰＨＯ８１蛋白的二级结构。用Ｋｙｔｅ－Ｄｏｏｌｉｔｔｌｅ方法分析它的疏水性。分析了ＰＨＯ８１蛋白与其他蛋白的同源性,发现它包含５个ＳＷ１６／ＡＮＫ重复单位,这些重复单位可能是ＰＨＯ８１蛋白与负调控因子ＰＨＯ８０相互作用的位点。     

Nuclear magnetic resonance assignment and secondary structure of an ankyrin-like repeat-bearing protein: myotrophin.

Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 11B amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two- and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly 15N-labeled or uniformly 15N/13C-labeled protein to obtain the 1H, 15N, and 13C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOEs, NH exchange data, 3JHN alpha coupling constants, and chemical shifts of 1H alpha, 13C alpha, and 13 C beta. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.     

Effects of host interaction with Wolbachia on cytoplasmic incompatibility in the two‐spotted spider mite Tetranychus urticae

The endosymbiotic bacterium Wolbachia pipientis infects a wide range of arthropods and induces a variety of reproductive anomalies, including cytoplasmic incompatibility (CI). Three populations of the two‐spotted spider mite, Tetranychus urticae, were investigated in the present study. Based on gene sequencing, they had different host genetic backgrounds but were infected with the same Wolbachia strain. We also examined the CI level relative to host background, male age, Wolbachia density, and Ankyrin (ANK) gene expression in T. urticae. The results of the present study suggest that: (1) CI differences between populations appear to be a result of host genetic background; (2) male age is not a factor determining intensity of CI; (3) Wolbachia density in males may serve as threshold factor necessary for the CI to occur in T. urticae, after which other factors become important in determining the strength of CI; and (4) hosts may modulate CI intensity through modulation of ANK gene expression in males. Our results describe a new type of interaction between Wolbachia and its hosts, and the effect of the interactions on CI. Further investigations on the functions of Wolbachia ankyrin gene products and their host targets, particularly with respect to host reproductive manipulation, are also imperative.