Robert A. Sikkink

Failure of Calcineurin Inhibitors to Prevent Pressure-Overload Left Ventricular Hypertrophy in Rats

A rapidly emerging body of literature implicates a pivotal role for the Ca2+-calmodulin-dependent phosphatase calcineurin as a cellular target for a variety of Ca2+-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. kg-1. d-1) 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. kg-1. d-1) or even higher doses of CsA (10 and 20 mg. kg-1. d-1) 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 pressure-overload hypertrophy can arise through calcineurin-independent pathways.

Identification of Gene Mutations in Autosomal Dominant Polycystic Kidney Disease through Targeted Resequencing

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 bar-coded 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.

Kinetic and spectroscopic analyses of mutants of a conserved histidine in the metallophosphatases calcineurin and lambda protein phosphatase.

Calcineurin belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage λ 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 λ protein phosphatase accommodates a dinuclear metal center. Calcineurin and λ 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 λ 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 λ protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease ink cat of 1,300-fold for calcineurin. With the analogous λ protein phosphatase mutant, k catdecreased 530-fold compared with wild type λ 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.

Mate Pair Sequencing of Whole-Genome-Amplified DNA Following Laser Capture Microdissection of Prostate Cancer

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.

Characterization of the calcium-binding sites of calcineurin B

Calcineurin (CaN) is a calcium‐ and calmodulin‐dependent serine/threonine phosphatase whose inhibition by the immunosuppressant‐immunophilin complexes (cyclosporin‐cyclophilin and FK506‐FKBP) is considered key to the mechanism of immunosuppression. CaN is a heterodimer, consisting of a 59 kDa catalytic subunit (A) and a 19 kDa calcium‐binding regulatory subunit (B). The latter is postulated to harbor four calcium binding domains of the EF hand type. The titration of the CaN B apoprotein with the isomorphic Cd2+ was followed by 113Cd NMR and these data support one high‐affinity metal binding site and three lower‐affinity ones. Flow dialysis data with Ca2+ indicate one high affinity calcium binding site with K d ∼ 2.4 × 10−8 M and three other sites with K d ∼ 1.5 × 10−5 M. The chemical shifts of all four 113Cd resonances (−75, −93, −106 and −119 ppm) are in the same range as found in other 113Cd substituted calcium‐binding proteins, and are indicative of all‐oxygen coordination of pentagonal bipyramidal geometry.

Contribution of neelaredoxin to oxygen tolerance by Treponema pallidum.

Publisher Summary The present in vitro cocultivation system allows investigators to monitor treponernal responses to various oxygen tensions. As such, syphilis researchers are now poised to compare both the transcriptional and enzymatic activities of the putative oxygen detoxification genes under controlled oxygen tensions. Conversely, applying these methodologies to treponemes harvested from rabbit tissues, blood, and cerebrospinal fluid could yield clues to the ability of the treponeme to survive hematogeneous dissemination and cerebrospinal fluid (CSF) invasion. Presumably, comparing transcriptional profiles of in vitro- and in vivo-grown treponemes would shed light on the critical “missing ingredients” of the current in vitro cultivation techniques. This chapter presents the methods of treponemal propagation both in vivo and in vitro , and the techniques employed to analyze the expression and enzymatic activity of neelaredoxin. A similar conceptual and methodological synthesis could be engaged to empirically confirm the identity of the remaining constituents of the oxygen detoxification pathway in this bacterium.

The use of 113Cd NMR chemical shifts as a structural probe in tetrathiolate metalloproteins

Abstract A wide range of 113Cd chemical shifts has been observed for 113Cd-substituted metalloproteins ranging from −100 ppm, for Cd with octahedral oxygen ligands, to +760 ppm for tetrahedral sulfur ligands. In particular the 113Cd chemical shifts of tetrahedral sulfur bound sites, for proteins such as rubredoxin and desulforedoxin, appear around 720–745 ppm. New 113Cd chemical shift data for 113Cd-substituted, overexpressed and mutated homologous desulforedoxin-like Fe(S-Cys)4 proteins, have been obtained and a correlation between the 113Cd chemical shift and structure at the metal site has been observed. This subtle effect of geometry at the metal centre on 113Cd chemical shifts can be explained in terms of an increase in the paramagnetic term for the chemical shift of the 113Cd nucleus as distortion of the tetrathiolate centre is increased.

Differential modulation of cortical synaptic activity by calcineurin (phosphatase 2B) versus phosphatases 1 and 2A

Reversible protein phosphorylation is thought to play an important regulatory role in synaptic neurotransmission. We recently have shown in cultured rat cortical neurons that inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin (phosphatase 2B) increases the frequency, but not the amplitude, of postsynaptic glutamatergic currents, implicating a presynaptic site of action for calcineurin. The specific presynaptic phosphoprotein substrates for calcineurin are unknown, however, calcineurin has been implicated in the control of the Ca2+-independent phosphatases, phosphatases 1 and 2A. To determine whether calcineurins effects on synaptic transmission are direct or are mediated by changes in phosphatase 1 and/or 2A activities, we used whole-cell voltage clamp to record spontaneous and miniature excitatory postsynaptic currents in the presence of calyculin A (1 microM in bath solution), a membrane permeant inhibitor of phosphatases 1 and 2A which has no effect on calcineurin. Calyculin increased postsynaptic current amplitude without changing current frequency. In these same neurons, subsequent inhibition of calcineurin with cyclosporine A or FK506 had no further effect on current amplitude, but increased current frequency. The increased current amplitude seen with calyculin involved a postsynaptic mechanism, since the effect was reproduced by microcystin (10 microM in pipette solution), which is a membrane-impermeant inhibitor of phosphatases 1 and 2A. Thus, in rat cortical neurons, glutamatergic neurotransmission appears to be frequency-modulated through a presynaptic mechanism by calcineurin, and amplitude-modulated through a postsynaptic mechanism by phosphatases 1 and 2A.

A simple method for gene phasing using mate pair sequencing

BackgroundRecessive genes cause disease when both copies are affected by mutant loci. Resolving the cis/trans relationship of variations has been an important problem both for researchers, and increasingly, clinicians. Of particular concern are patients who have two heterozygous disease-causing mutations and could be diagnosed as affected (one mutation on each allele) or as phenotypically normal (both mutations on the same allele). Several methods are currently used to phase genes, however due to cost, complexity and/or low sensitivity they are not suitable for clinical purposes.MethodsLong-range amplification was used to select and enrich the target gene (CYP21A2) followed by modified mate-pair sequencing. Fragments that mapped coincidently to two heterozygous sites were identified and used for statistical analysis.ResultsProbabilities for cis/trans relationships between heterozygous positions were calculated along with 99% confidence intervals over the entire length of our 10 kb amplicons. The quality of phasing was closely related to the depth of coverage and the number of erroneous reads. Most of the error was found to have been introduced by recombination in the PCR reaction.ConclusionsWe have developed a simple method utilizing massively parallel sequencing that is capable of resolving two alleles containing multiple heterozygous positions. This method stands out among other phasing tools because it provides quantitative results allowing confident haplotype calls.

Clinical Validation of a Haplotyping Method with Next-Generation Sequencing

To the Editor: One of the most persistent problems in clinical genetic testing is interpretation of compound heterozygotes. Particularly problematic are recessive genes for which compound heterozygosity is relatively common, such as CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), the gene responsible for >90% of congenital adrenal hyperplasia (CAH)1 cases. Next-generation sequencing (NGS) methods provide a way to both sequence and phase genes; however, the techniques are largely qualitative and do not provide a quantitative measure of confidence in the cis/trans call. To date, an intuitive and reliable statistic to summarize the quality of a phase call is not available for diagnostic sequencing. Without such an indicator, incorrect phase calls can be made easily when sequence coverage is low or when errors are introduced during library preparation or amplification. Previously, we described a statistical method to generate probability scores and associated confidence intervals for each base in a tandem sequence of heterozygous positions (1). That method is based on stepwise analysis of sequential pairs of heterozygous loci. Although the method is functional, it suffers in regions where coverage is low and …