Mutations in two large multi-exon genes, PKD1 and PKD2, cause autosomal dominant polycystic kidney disease (ADPKD). The duplication of PKD1 exons 1-32 as six pseudogenes on chromosome 16, the high level of allelic heterogeneity, and the cost of Sanger sequencing complicate mutation analysis, which can aid diagnostics of ADPKD. We developed and validated a strategy to analyze both the PKD1 and PKD2 genes using next-generation sequencing by pooling long-range PCR amplicons and multiplexing barcoded libraries. We used this approach to characterize a cohort of 230 patients with ADPKD. This process detected definitely and likely pathogenic variants in 115 (63%) of 183 patients with typical ADPKD. In addition, we identified atypical mutations, a gene conversion, and one missed mutation resulting from allele dropout, and we characterized the pattern of deep intronic variation for both genes. In summary, this strategy involving next-generation sequencing is a model for future genetic characterization of large ADPKD populations.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa ␣ 2 homodimer containing, on average, 0.7 iron atoms/subunit. Mö ssbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K 3 (Fe(CN) 6 ), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A 280 /A 656 ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

Calcineurin belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage protein phosphatase is also considered to be a member of this family based on sequence comparisons (Lohse, D. L., Denu, J. M., and Dixon, J. E. (1995) Structure 3, 987-990). Using EPR spectroscopy, we demonstrate that protein phosphatase accommodates a dinuclear metal center. Calcineurin and protein phosphatase likewise contain a conserved histidine that is not a metal ligand but is within 5 Å of either metal in calcineurin. In this study the conserved histidine in calcineurin was mutated to glutamine and the mutant protein analyzed by EPR spectroscopy and kinetic methods. Parallel studies with an analogous protein phosphatase mutant were also carried out. Kinetic studies using paranitrophenyl phosphate as substrate showed a decrease in k cat of 460-and 590-fold for the calcineurin and protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease in k cat of 1,300-fold for calcineurin. With the analogous protein phosphatase mutant, k cat decreased 530-fold compared with wild type protein phosphatase using phenyl phosphate as a substrate. EPR studies of the iron-reconstituted enzymes indicated that although both mutant enzymes can accommodate a dinuclear metal center, spectroscopic differences compared with wild type proteins suggest a perturbation of the ligand environment, possibly by disruption of a hydrogen bond between the histidine and a metal-coordinated solvent molecule.Calcineurin, also known as protein phosphatase 2B, consists of a 58-kDa catalytic subunit, calcineurin A, and a 19-kDa regulatory subunit, calcineurin B. It is a serine/threonine protein phosphatase whose activity is regulated by Ca 2ϩ /calmodulin. Calcineurin is the target of the immunosuppressant drugs cyclosporin A and FK506 (1, 2). These drugs bind to intracellular proteins, termed immunophilins; cyclophilin is the binding protein for cyclosporin A, and FK506 binds to the FK506-binding proteins. The complex of immunosuppressant drug and immunophilin in turn binds to and inhibits the phosphatase activity of calcineurin. Calcineurin inhibition prevents the transcriptional activation of the interleukin 2 gene in helper T cells, leading to suppression of the immune response.Calcineurin is a member of the class of serine/threonine protein phosphatases, whose members include protein phosphatases 1 (PP1) 1 and 2A (PP2A), phosphatases essential for a number of signal transduction pathways in eukaryotic cells (3, 4). Another protein phosphatase from bacteriophage , PP, also belongs to this family (5). In addition, a number of less characterized enzymes containing the "phosphoesterase" consensus motif of this family, DXH(X) n GDXXD(X) n GNHD/E, have been identified via protein sequence comparisons (6, 7). It has been hypothesized that this motif provides a scaffold for an active site dinuclear meta...

Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM)

Calcineurin, a Ca 2؉ /calmodulin-stimulated protein phosphatase, plays a key role in T-cell activation by regulating the activity of NFAT (nuclear factor of activated T cells), a family of transcription factors required for the synthesis of several cytokine genes. Calcineurin is the target of the immunosuppressive drugs cyclosporin A and FK506 complexed with their cytoplasmic receptors cyclophilin and FKBP12, respectively. In this study we report that calcineurin is also the target of a recently identified Ca 2؉ -binding protein, CHP (for calcineurin homologous protein), which shares a high degree of homology with the regulatory B subunit of calcineurin and with calmodulin. In Jurkat and HeLa cells, overexpression of CHP specifically impaired the nuclear translocation and transcriptional activity of NFAT but had no effect on AP-1 transcriptional activity and only a small (<25%) inhibitory effect on the transcriptional activity of NFB. Further study indicated that CHP inhibits calcineurin activity. In cells overexpressing CHP, the phosphatase activity of immunoprecipitated calcineurin was inhibited by ϳ50%; and in a reconstituted assay, the activity of purified calcineurin was inhibited up to 97% by the addition of purified recombinant CHP in a dose-dependent manner. Moreover, prolonged activation of Jurkat cells was associated with a decreased abundance of CHP, suggesting a possible regulatory mechanism allowing activation of calcineurin. CHP, therefore, is a previously unrecognized endogenous inhibitor of calcineurin activity.Stimulation of T cells results in the induction of several cytokine genes that are involved in the control of T-cell proliferation and immune responses (1-3). One of the signaling pathways mediating T-cell receptor activation involves the elevation of intracellular calcium ([Ca 2ϩ ] i ) and the activation of a key signaling enzyme, calcineurin (4, 5). A rise in [Ca 2ϩ ] i leads to the activation of calcineurin and the dephosphorylation and nuclear translocation of NFAT (nuclear factor of activated T cells).1 This in turn drives transcription of response genes such as the cytokines interleukin (IL)-2 and IL-4 and the CD44 ligand (6 -11). Calcineurin is a heterodimeric enzyme consisting of a catalytic A subunit (CnA) and a tightly associated Ca 2ϩ -binding regulatory B subunit (CnB) (12, 13). Maximal calcineurin phosphatase activity requires the Ca 2ϩ -dependent binding of calmodulin (CaM) to the CnA-CnB complex at a domain independent of the CnB-binding site on CnA (14).We recently identified a novel, ubiquitously expressed, Ca 2ϩ -binding protein, CHP (for calcineurin homologous protein), which shares a high degree of similarity with CnB (65%) and CaM (59%) (15). Although CHP was originally identified by screening a library to detect proteins that interacted with the Na ϩ /H ϩ exchanger isoform NHE1, subsequent studies indicated that CHP has actions that are independent of its regulation of NHE1. Overexpression of CHP in mutant NHE-deficient fibroblasts inhibits cell proliferation, ...

Abstract-A rapidly emerging body of literature implicates a pivotal role for the Ca 2ϩ -calmodulin-dependent phosphatase calcineurin as a cellular target for a variety of Ca 2ϩ -dependent signaling pathways culminating in left ventricular hypertrophy (LVH). Most of the recent experimental support for this hypothesis is derived from in vitro studies or in vivo studies in transgenic mice expressing activated calcineurin or mutant sarcomeric proteins. The aim of the present study was to test whether calcineurin inhibitors, cyclosporin A (CsA) and FK 506, prevent pressure-overload LVH using 2 standard rat models: (1) the spontaneously hypertensive rat (SHR) and (2) aortic banding. The major new findings are 2-fold. First, in SHR, LVH (left ventricular weight to body weight ratio) was unaffected by a dose of CsA (5 mg ⅐ kg Ϫ1 ⅐ d Ϫ1) that was sufficient to raise blood pressure and to inhibit calcineurin-mediated transcriptional activation in skeletal muscle. Second, in rats with aortic banding, LVH was unaffected by FK 506 (0.3 mg ⅐ kg Ϫ1 ⅐ d Ϫ1 ) or even higher doses of CsA (10 and 20 mg ⅐ kg) that were sufficient to inhibit 90% of total calcineurin phosphatase activity in the hypertrophied myocardium. In the latter experiments, CsA blocked neither the elevated left ventricular end-diastolic pressures, a measure of diastolic function, nor the induction in atrial natriuretic peptide mRNA in the hypertrophic ventricles. Thus, in numerous experiments, systemic administration of potent calcineurin inhibitors did not prevent the development of LVH in 2 classic models of pressure-overload hypertrophy. These results demonstrate that pressureoverload hypertrophy can arise through calcineurin-independent pathways. (Circ Res. 1999;84:722-728.) Key Words: left ventricular hypertrophy Ⅲ calcineurin Ⅲ cyclosporin A Ⅲ spontaneously hypertensive rat Ⅲ aortic banding I n patients with many forms of heart disease, the development of left ventricular hypertrophy (LVH) constitutes one of the most powerful predictors of subsequent cardiovascular morbidity and mortality. 1-3 Increased left ventricular mass strongly predicts an increased incidence of cardiovascular death, myocardial infarction, angina requiring revascularization, stroke, and congestive heart failure. Despite the clinical importance of LVH, the underlying mechanisms mediating the hypertrophic process have been enigmatic.A variety of intrinsic and extrinsic pathological stimuli can initiate a hypertrophic response in the left ventricle. Extrinsic stimuli include pressure overload, volume overload, and neurohumoral factors. Intrinsic stimuli include contractile abnormalities resulting from altered expression or mutations of sarcomeric proteins. Although these diverse stimuli are known to activate diverse intracellular signal transduction pathways, 4 it is unknown whether these various pathways ultimately converge on a final common signaling pathway culminating in cardiomyocyte hypertrophy. 4 -6 In this regard, changes in [Ca 2ϩ ] i have been proposed to play a pivotal rol...

High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.

Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.