Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways.

Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.

WNKs are potassium-sensitive kinases.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.

A Phosphorylated Intermediate in the Activation of WNK Kinases.

WNK kinases autoactivate by autophosphorylation. Crystallography of the kinase domain of WNK1 phosphorylated on the primary activating site (pWNK1) in the presence of AMP-PNP reveals a well-ordered but inactive configuration. This new pWNK1 structure features specific and unique interactions of the phosphoserine, less hydration, and smaller cavities compared with those of unphosphorylated WNK1 (uWNK1). Because WNKs are activated by osmotic stress in cells, we addressed whether the structure was influenced directly by osmotic pressure. pWNK1 crystals formed in PEG3350 were soaked in the osmolyte sucrose. Suc-WNK1 crystals maintained X-ray diffraction, but the lattice constants and pWNK1 structure changed. Differences were found in the activation loop and helix C, common switch loci in kinase activation. On the basis of these structural changes, we tested for effects on in vitro activity of two WNKs, pWNK1 and pWNK3. The osmolyte PEG400 enhanced ATPase activity. Our data suggest multistage activation of WNKs.

Newborn screening and single nucleotide variation profiling of TSHR, TPO, TG and DUOX2 candidate genes for congenital hypothyroidism.

High prevalence of congenital hypothyroidism (CH) among Indian newborns prompted us to establish population-specific reference ranges of TSH and to explore the contribution of the common genetic variants in TSHR, TPO, TG and DUOX2 genes towards CH. A total of 1144 newborns (593 males and 551 females) were screened for CH. SNV profiling (n = 22) spanning three candidate genes, i.e. TSHR, TPO and TG was carried out in confirmed CH cases (n = 45). In screen negative cases (n = 700), ten TSHR variants were explored to establish association with CH. No mutation found in DUOX2. The 2.5th to 97.5th percentiles of TSH in these newborns were 0.5 to 12.2 mU/L. In newborns with optimal birth weight, the cut-off was 10 mU/L. Lower or higher birth weight resulted in slightly higher TSH. Two TSHR variants, i.e. rs7144481 and rs17630128 were associated with agenesis, hypoplasia and goiter. The rs2268477 was associated with agenesis and hypoplasia. The rs1991517, rs2075176 and rs2241119 were associated with agenesis only. The rs7144481, rs17630128, rs1991517 and rs2268477 were associated with 2.17, 4.62, 2.91 and 2.29-fold increased risk for CH, respectively. Among the TPO variants, rs867983 and rs2175977 were associated with agenesis and goiter, respectively. Among the TG variants, rs2076740 showed association with agenesis and goiter. Two rare variants i.e. TPO g.IVS14-19 G>C and TG c.1262 C>T were observed in CH cases. No genetic variant identified in the two exons of DUOX2. To conclude, the current study established Indian population-specific normative values for TSH and demonstrates specific genotype-phenotype correlations among three candidate genes.

Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule.

Background With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule.Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux.ResultsIn vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, Drosophila WNK activity acutely increased. Drosophila WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux.Conclusions Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

Spectrum of SMPD1 mutations in Asian-Indian patients with acid sphingomyelinase (ASM)-deficient Niemann-Pick disease.

Acid sphingomyelinase (ASM)-deficient Niemann-Pick disease is an autosomal recessive lysosomal storage disorder caused by biallelic mutations in the SMPD1 gene. To date, around 185 mutations have been reported in patients with ASM-deficient NPD world-wide, but the mutation spectrum of this disease in India has not yet been reported. The aim of this study was to ascertain the mutation profile in Indian patients with ASM-deficient NPD. We sequenced SMPD1 in 60 unrelated families affected with ASM-deficient NPD. A total of 45 distinct pathogenic sequence variants were found, of which 14 were known and 31 were novel. The variants included 30 missense, 4 nonsense, and 9 frameshift (7 single base deletions and 2 single base insertions) mutations, 1 indel, and 1 intronic duplication. The pathogenicity of the novel mutations was inferred with the help of the mutation prediction software MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using the structure modeled with the help of the SWISS-MODEL workspace program. The p. (Arg542*) (c.1624C>T) mutation was the most commonly identified mutation, found in 22% (26 out of 120) of the alleles tested, but haplotype analysis for this mutation did not identify a founder effect for the Indian population. To the best of our knowledge, this is the largest study on mutation analysis of patients with ASM-deficient Niemann-Pick disease reported in literature and also the first study on the SMPD1 gene mutation spectrum in India. © 2016 Wiley Periodicals, Inc.

Discovery of novel TAOK2 inhibitor scaffolds from high-throughput screening.

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.

Precisely ordered phosphorylation reactions in the p38 mitogen-activated protein (MAP) kinase cascade.

The MAP kinase cascades, composed of a MAP3K, a MAP2K, and a MAPK, control switch responses to extracellular stimuli and stress in eukaryotes. The most important feature of these modules is thought to be the two double phosphorylation reactions catalyzed by MAP3Ks and MAP2Ks. We addressed whether the reactions are sequential or random in the p38 MAP kinase module. Mass spectrometry was used to track the phosphorylation of the MAP2K MEK6 by two MAP3Ks, TAO2 and ASK1, and the subsequent phosphorylation of p38α by MEK6/S*T* (where S (Ser) and T (Thr) are the two phosphorylation sites and * denotes phosphorylation). Both double phosphorylation reactions are precisely ordered. MEK6 is phosphorylated first on Thr-211 and then on Ser-207 by both MAP3Ks. This is the first demonstration of a precise reaction order for a MAP2K. p38α is phosphorylated first on Tyr-182 and then on Thr-180, the same reaction order observed previously in ERK2. Thus, intermediates were MEK6/ST* and p38α/TY*. Similarly, the phosphorylation of the p38α transcription factor substrate ATF2 occurs in a precise sequence. Progress curves for the appearance of intermediates were fit to kinetic models. The models confirmed the reaction order, revealed processivity in the phosphorylation of MEK6 by ASK1, and suggested that the order of phosphorylation is dictated by both binding and catalysis rates.

Adaptive developmental plasticity in methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism limits its frequency in South Indians.

Methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism shows considerable heterogeneity in its distribution in humans worldwide. The current study was conducted to investigate whether this polymorphism exhibited adaptive developmental plasticity in the control of the TT-genotype frequency. We screened 1,818 South Indian subjects (895 males and 923 females) for MTHFR C677T polymorphism using PCR-restriction fragment length polymorphism approach. MTHFR 677T-allele frequency in males and females was 9.1 and 11.0%, respectively. Compared to females, males had lower frequency of TT-genotype [odds ratio 0.31, 95% confidence interval (CI) 0.08-1.01]. The frequency of MTHFR 677T-allele was highest in the age group of 20-40 years and it gradually decreased from 40-60 to 60-80 years (P trend<0.0001). MTHFR 677TT-genotype was associated with 7.02-folds (95% CI: 2.12-25.63, P<0.0001) cumulative risk for recurrent pregnancy loss (RPL), neural tube defects (NTDs) and deep vein thrombosis (DVT). Linear regression model suggested that male gender exhibited increased homocysteine levels by 9.35 µmol/L while each MTHFR 677T-allele contributed to 4.63 µmol/L increase in homocysteine. Plasma homocysteine showed inverse correlation with dietary folate (r=-0.17, P<0.0001), B2 (r=-0.14, P<0.0001) and B6 (r=-0.07, P=0.03). Examination of the spontaneously aborted fetuses (n=35) showed no significant association of fetal genotype on its in utero viability. From the current study, it was concluded that C677T seemed to have acquired adaptive developmental plasticity among South Indians due to environmental influences thus contributing to hyperhomocysteinemia and its associated complications such as RPL, NTDs, DVT, etc.

Autistic children exhibit distinct plasma amino acid profile.

In order to ascertain whether autistic children display characteristic metabolic signatures that are of diagnostic value, plasma amino acid analyses were carried out on a cohort of 138 autistic children and 138 normal controls using reverse-phase HPLC. Pre-column derivatization of amino acids with phenyl isothiocyanate forms phenyl thio-carbamate derivates that have a lamba(max) of 254 nm, enabling their detection using photodiode array. Autistic children showed elevated levels of glutamic acid (120 +/- 89 vs. 83 +/- 35 micromol/L) and asparagine (85 +/- 37 vs. 47 +/- 19 micromol/L); lower levels of phenylalanine (45 +/- 20 vs. 59 +/- 18 micromol/L), tryptophan (24 +/- 11 vs. 41 +/- 16 micromol/L), methionine (22 +/- 9 vs. 28 +/- 9 micromol/L) and histidine (45 +/- 21 vs. 58 +/- 15 micromol/L). A low molar ratio of (tryptophan/large neutral amino acids) x 100 was observed in autism (5.4 vs 9.2), indicating lesser availability of tryptophan for neurotransmitter serotonin synthesis. To conclude, elevated levels of excitatory amino acids (glutamate and asparagine), decreased essential amino acids (phenylalanine, tryptophan and methionine) and decreased precursors of neurotransmitters (tyrosine and tryptophan) are the distinct characteristics of plasma amino acid profile of autistic children. Thus, such metabolic signatures might be useful tools for early diagnosis of autism.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing.

Previous observations showed that chloride and osmotic stress regulate the autophosphorylation and activity of the kinase domains of WNK1 and WNK3. Further, prior crystallography on the asymmetric dimeric of the unphosphorylated WNK1 kinase domain (WNK1/S382A, WNK1/SA) revealed conserved waters in the active site. Here we show by crystallography that PEG400 applied to crystals of dimeric WNK1/SA grown in space group P1 induces de-dimerization with a change in space group to P2 1 . Both the conserved waters, referred to here as conserved water network 1 (CWN1) and the chloride binding site are disrupted by PEG400. CWN1 is surrounded and stabilized by a pan-WNK-conserved cluster of charged residues. Here we mutagenized these charges in WNK3 to probe the importance of the CWN1 to WNK regulation. Two mutations at E314 in the Activation Loop (WNK3/E314Q and WNK3/E314A) enhanced activity, consistent with the idea that the CWN1 is inhibitory. Mutations of other residues in the cluster had similar or less activity than wild-type. PEG400 activation of WNK3 was not significantly reduced in the point mutants tested. The crystallographic and assay data support a role for CWN1 and the charged cluster in stabilizing an inactive configuration of WNKs and suggest that water functions as an allosteric inhibitor of WNKs.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

Hydrostatic Pressure Sensing by WNK kinases.

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

3-M syndrome - a primordial short stature disorder with novel CUL7 mutation in two Indian patients.

OBJECTIVE: To evaluate the cause of short stature in children. CASE PRESENTATION: Two children with suspected skeletal dysplasia and short stature were evaluated. CONCLUSIONS: The 3-M syndrome is a primordial growth disorder manifesting severe postnatal growth restriction, skeletal anomalies and prominent fleshy heels. The 3-M syndrome is a genetically heterogeneous disorder and the phenotype is similar. This is a rare autosomal recessive disorder with normal intellect. Two affected children have been identified by whole-exome sequencing. One patient harboured a compound heterozygous variant and the other was a homozygous missense variant. The genetic diagnosis helped in counselling the families and facilitated prenatal diagnosis in one (case 1) family.

Synthesis and Structural Characterization of Novel Trihalo-sulfone Inhibitors of WNK1.

With No lysine (K) [WNK] kinases are structurally unique serine/threonine protein kinases that have therapeutic potential for blood pressure regulation and cancer. A novel class of trihalo-sulfone compounds was identified by high-throughput screening. Trihalo-sulfone 1 emerged as an effective inhibitor of WNK1 with an IC50 value of 1.6 µM. Herein, we define chemical features necessary for inhibition of WNK1 using chemical synthesis and X-ray crystallography. Analogues that probed the role of specific functional groups to the inhibitory activity were synthesized. X-ray structures of trihalo-sulfone 1 and a second trihalo-sulfone 23 bound to WNK1 revealed active site binding to two of the three previously defined canonical inhibitor binding pockets as well as a novel binding site for the trihalo-sulfone moiety. The elucidation of these novel interaction sites may allow for the strategic design of even more selective and potent WNK inhibitors.

Cerebral creatine deficiency disorders - A clinical, genetic and follow up study from India.

INTRODUCTION: Cerebral creatine deficiency syndromes (CCDS) are a group of potentially treatable neurometabolic disorders. The clinical, genetic profile and follow up outcome of Indian CCDS patients is presented. MATERIALS AND METHODS: This was a retrospective cohort of CCDS patients seen over six-years. Diagnosis was based either on low creatine peak on proton magnetic resonance spectroscopy (MRS) and/or genetic evaluation. RESULTS: Thirteen patients were eligible [8 creatine transporter deficiency (CTD), 4 guanidinoacetate methyltransferase (GAMT) deficiency and 1 could not be classified]. The mean (±SD) age at diagnosis was 7.2(±5.0) years. Clinical manifestations included intellectual disability (ID) with significant expressive speech delay in all. Most had significant behavior issues (8/13) and/or autism (8/13). All had history of convulsive seizures (11/13 had epilepsy; 2 patients only had febrile seizures) and 2/13 had movement disorder. Constipation was the commonest non-neurological manifestation (5/13 patients). Cranial MRI was normal in all CTD patients but showed globus pallidus hyperintensity in all four with GAMT deficiency. MRS performed in 11/13 patients, revealed abnormally low creatine peak. A causative genetic variant (novel mutation in nine) was identified in 12 patients. Three GAMT deficiency and one CTD patient reported neurodevelopmental improvement and good seizure control after creatine supplementation. CONCLUSION: Intellectual disability, disproportionate speech delay, autism, and epilepsy, were common in our CCDS patients. A normal structural neuroimaging with easily controlled febrile and/or afebrile seizures differentiated CTD from GAMT deficiency patients who had abnormal neuroimaging and often difficult to control epilepsy and movement disorder.

The structure of the MAP2K MEK6 reveals an autoinhibitory dimer.

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/DeltaN/DD) has been solved at 2.3 angstroms resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/DeltaN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare "arginine stack" between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.

Molecular insights into the role of genetic determinants of congenital hypothyroidism.

In our previous studies, we have demonstrated the association of certain variants of the thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (TG) genes with congenital hypothyroidism. Herein, we explored the mechanistic basis for this association using different in silico tools. The mRNA 3'-untranslated region (3'-UTR) plays key roles in gene expression at the post-transcriptional level. In TSHR variants (rs2268477, rs7144481, and rs17630128), the binding affinity of microRNAs (miRs) (hsa-miR-154-5p, hsa-miR-376a-2-5p, hsa-miR-3935, hsa-miR-4280, and hsa-miR-6858-3p) to the 3'-UTR is disrupted, affecting post-transcriptional gene regulation. TPO and TG are the two key proteins necessary for the biosynthesis of thyroid hormones in the presence of iodide and H2O2. Reduced stability of these proteins leads to aberrant biosynthesis of thyroid hormones. Compared to the wild-type TPO protein, the p.S398T variant was found to exhibit less stability and significant rearrangements of intra-atomic bonds affecting the stoichiometry and substrate binding (binding energies, ΔG of wild-type vs. mutant: ‒15 vs. ‒13.8 kcal/mol; and dissociation constant, Kd of wild-type vs. mutant: 7.2E-12 vs. 7.0E-11 M). The missense mutations p.G653D and p.R1999W on the TG protein showed altered ΔG (0.24 kcal/mol and 0.79 kcal/mol, respectively). In conclusion, an in silico analysis of TSHR genetic variants in the 3'-UTR showed that they alter the binding affinities of different miRs. The TPO protein structure and mutant protein complex (p.S398T) are less stable, with potentially deleterious effects. A structural and energy analysis showed that TG mutations (p.G653D and p.R1999W) reduce the stability of the TG protein and affect its structure-functional relationship.

Osmosensing by WNK Kinases.

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

Role of parental folate pathway single nucleotide polymorphisms in altering the susceptibility to neural tube defects in South India.

AIM: To investigate the role of four parental folate pathway single nucleotide polymorphisms (SNPs) i.e., methylene tetrahydrofolate reductase (MTHFR) 677C>T, MTHFR 1298A>C, methionine synthase reductase (MTRR) 66A>G and glutamate carboxypeptidase (GCP) II 1561C>T on susceptibility to neural tube defects (NTDs) in 50 couples with NTD offspring and 80 couples with normal pregnancy outcome. RESULTS: Maternal MTHFR 677C-->T (odds ratio (OR): 2.69, 95% confidence interval (CI): 1.35-5.34) and parental GCP II 1561C-->T (maternal: OR: 1.89, 95% CI: 1.12-3.21 and paternal: OR: 3.23, 95% CI: 1.76-5.93) were found to be risk factors for a NTD. Both paternal and maternal GCP II T-variant alleles were found to interact with MTHFR 677T- and MTRR G-variant alleles in increasing the risk for NTD. Segregation of data based on type of defect revealed an association between maternal 677T-allele and meningomyelocele (OR: 9.00, 95% CI: 3.77-21.55, P<0.0001) and an association between parental GCP II 1561T-allele and anencephaly (maternal: OR: 2.25, 95% CI: 1.12-4.50, P<0.05 and paternal: OR: 4.26, 95% CI: 2.01-9.09, P<0.001). CONCLUSIONS: Maternal MTHFR C677T and parental GCP II C1561T polymorphisms are associated with increased risk for NTDs. Apart from individual genetic effects, epistatic interactions were also observed.