The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin.

Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca(2+)/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, "LxVP." The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that "LxVP" sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent "LxVP"-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.

TFEB drives mTORC1 hyperactivation and kidney disease in Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, leading to hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and lesions  in multiple organs including lung (lymphangioleiomyomatosis) and kidney (angiomyolipoma and renal cell carcinoma). Previously, we found that TFEB is constitutively active in TSC. Here, we generated two mouse models of TSC in which kidney pathology is the primary phenotype. Knockout of TFEB rescues kidney pathology and overall survival, indicating that TFEB is the primary driver of renal disease in TSC. Importantly, increased mTORC1 activity in the TSC2 knockout kidneys is normalized by TFEB knockout. In TSC2-deficient cells, Rheb knockdown or Rapamycin treatment paradoxically increases TFEB phosphorylation at the mTORC1-sites and relocalizes TFEB from nucleus to cytoplasm. In mice, Rapamycin treatment normalizes lysosomal gene expression, similar to TFEB knockout, suggesting that Rapamycin's benefit in TSC is TFEB-dependent. These results change the view of the mechanisms of mTORC1 hyperactivation in TSC and may lead to therapeutic avenues.

Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation.

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases ( approximately 50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.

MethylMasteR: A Comparison and Customization of Methylation-Based Copy Number Variation Calling Software in Cancers Harboring Large Scale Chromosomal Deletions.

DNA methylation-based copy number variation (CNV) calling software offers the advantages of providing both genetic (copy-number) and epigenetic (methylation) state information from a single genomic library. This method is advantageous when looking at large-scale chromosomal rearrangements such as the loss of the short arm of chromosome 3 (3p) in renal cell carcinoma and the codeletion of the short arm of chromosome 1 and the long arm of chromosome 19 (1p/19q) commonly seen in histologically defined oligodendrogliomas. Herein, we present MethylMasteR: a software framework that facilitates the standardization and customization of methylation-based CNV calling algorithms in a single R package deployed using the Docker software framework. This framework allows for the easy comparison of the performance and the large-scale CNV event identification capability of four common methylation-based CNV callers. Additionally, we incorporated our custom routine, which was among the best performing routines. We employed the Affymetrix 6.0 SNP Chip results as a gold standard against which to compare large-scale event recall. As there are disparities within the software calling algorithms themselves, no single software is likely to perform best for all samples and all combinations of parameters. The employment of a standardized software framework via creating a Docker image and its subsequent deployment as a Docker container allows researchers to efficiently compare algorithms and lends itself to the development of modified workflows such as the custom workflow we have developed. Researchers can now use the MethylMasteR software for their methylation-based CNV calling needs and follow our software deployment framework. We will continue to refine our methodology in the future with a specific focus on identifying large-scale chromosomal rearrangements in cancer methylation data.

Quantity of viable myocardium required to improve survival with revascularization in patients with ischemic cardiomyopathy: A meta-analysis.

BACKGROUND: This meta-analysis was conducted to determine optimal cutoff values for the assessment of viability using various imaging techniques for which revascularization would offer a survival benefit in patients with ischemic cardiomyopathy (ICM). METHODS AND RESULTS: We searched five electronic databases to identify relevant studies through December 2008. Relative risks of cardiac death, both in patients with and without viability, were calculated in each study. In order to estimate the optimal threshold for the presence of viability, we assumed a linear relationship between the amount of viable myocardium and survival benefit of revascularization. Twenty-nine studies (4,167 patients) met the inclusion criteria. The optimal threshold for the presence of viability was estimated to be 25.8% (95% CI: 16.6-35.0%) by positron emission tomography using 18F-fluorodeoxyglucose-perfusion mismatch, 35.9% (95% CI: 31.6-40.3%) by stress echocardiography using contractile reserve or ischemic responses, and 38.7% (95% CI: 27.7-49.7%) by single photon emission computed tomography using thallium-201 or technetium-99m MIBI myocardial perfusion. CONCLUSIONS: The calculated amount of viable myocardium determined to lead to improved survival was different among imaging techniques. Thus, separate cutoff values for imaging modalities may be helpful in determining which patients with ICM benefit from revascularization.

Carotid plaque, compared with carotid intima-media thickness, more accurately predicts coronary artery disease events: a meta-analysis.

OBJECTIVES: We conducted the meta-analysis to compare the diagnostic accuracies of carotid plaque and carotid intima-media thickness (CIMT) measured by B-mode ultrasonography for the prediction of coronary artery disease (CAD) events. METHODS: Two reviewers independently searched electronic databases to identify relevant studies through April 2011. Both population-based longitudinal studies with the outcome measure of myocardial infarction (MI) events and diagnostic cohort studies for the detection of CAD were identified and analyzed separately. Weighted summary receiver-operating characteristic (SROC) plots, with pertinent areas under the curves (AUCs), were constructed using the Moses-Shapiro-Littenberg model. Meta-regression analyses, using parameters of relative diagnostic odds ratio (DOR), were conducted to compare the diagnostic performance after adjusting other study-specific covariates. RESULTS: The meta-analysis of 11 population-based studies (54,336 patients) showed that carotid plaque, compared with CIMT, had a significantly higher diagnostic accuracy for the prediction of future MI events (AUC 0.64 vs. 0.61, relative DOR 1.35; 95%CI 1.1-1.82, p=0.04). The 10-year event rates of MI after negative results were lower with carotid plaque (4.0%; 95% CI 3.6-4.7%) than with CIMT (4.7%; 95% CI 4.2-5.5%). The meta-analysis of 27 diagnostic cohort studies (4.878 patients) also showed a higher, but non-significant, diagnostic accuracy of carotid plaque compared with CIMT for the detection of CAD (AUC 0.76 vs. 0.74, p=0.21 for relative DOR). CONCLUSIONS: The present meta-analysis showed that the ultrasound assessment of carotid plaque, compared with that of CIMT, had a higher diagnostic accuracy for the prediction of future CAD events.

Prediction of future cardiovascular outcomes by flow-mediated vasodilatation of brachial artery: a meta-analysis.

BACKGROUND: We conducted a meta-analysis of observational studies which examined the association between flow-mediated dilatation (FMD) of brachial artery, a noninvasive measure of endothelial function, and future cardiovascular events. METHODS: Electronic databases were searched using a predefined search strategy. Data was independently abstracted on study characteristics, study quality, and outcomes by two reviewers. The multivariate relative risks, adjusted for confounding factors, were calculated from individual studies and then pooled using random-effects models. Statistical heterogeneity was evaluated using I2 statistics. Subgroup analyses and meta-regression analyses were conducted to assess the robustness of the meta-analysis. Publication bias was examined with funnel plot analysis and Egger's test. RESULTS: Four population-based cohort studies and ten convenience-cohort studies, involving 5,547 participants, were included in the meta-analysis. The pooled relative risks of cardiovascular events per 1% increase in brachial FMD, adjusted for confounding risk factors, was 0.87 (95% CI, 0.83- 0.91). The significant associations between brachial FMD and cardiovascular events were consistent among all subgroups evaluated, suggesting the robustness of the meta-analysis. However, the presence of heterogeneity in study quality, the remaining confounding factors, and publication bias in the available literature prevent a definitive evaluation of the additional predictive value of brachial FMD beyond traditional cardiovascular risk factors. CONCLUSIONS: The meta-analysis of heterogeneous studies with moderate methodological quality suggested that impairment of brachial FMD is significantly associated with future cardiovascular events. Further prospective randomized trials are warranted to confirm the efficacy of the usage of brachial FMD in the management of cardiovascular diseases.

Impact of single or multicentre study design on the results of trials examining the efficacy of adjunctive devices to prevent distal embolisation during acute myocardial infarction.

AIMS: We investigated using meta-analytic techniques, whether, and to what degree, single or multicentre study design affects clinical outcomes in randomised controlled trials examining the efficacy of adjunctive devices to prevent distal embolisation during acute myocardial infarction (AMI). METHODS AND RESULTS: We searched electronic databases, conference proceedings, and internet-based sources of information to identify relevant studies through March 2009. The pooled summary effect was estimated with a random effects model. Subgroup and meta-regression analyses were conducted to examine the impact of single or multicentre design on trial outcomes compared with other variables. A total of 25 randomised trials (5,919 patients) were included in the analysis. The major sources of heterogeneity in trial outcomes were single or multicentre design, type of device used, study size, study region, and presence of conflicts of interest, of which the most influential source of heterogeneity was single or multicentre design (p-values of regression coefficient on meta-regression analyses were 0.09 for mortality, 0.001 for incomplete ST-segment resolution, and 0.07 for impaired myocardial blush grade, respectively). CONCLUSIONS: Single or multicentre study design has a significant impact on outcomes in trials examining the efficacy of adjunctive devices in AMI.

Ydj1 protects nascent protein kinases from degradation and controls the rate of their maturation.

Ydj1 is a Saccharomyces cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important for maintaining steady-state levels of protein kinases after screening several chaperones and cochaperones in gene deletion mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Delta mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Delta mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared to the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40, was unable to suppress defects in kinase accumulation in the ydj1Delta mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity. Our results suggest that Ydj1 can both protect nascent chains against degradation and control the rate of kinase maturation.