Doctor of Medicine, University of Michigan Ann Arbor (2007)
Bachelor of Arts, Pomona College (2001)
Jeffrey Feinstein, Postdoctoral Faculty Sponsor
Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.
View details for DOI 10.1172/JCI65592
View details for PubMedID 23867624
Protein phosphorylation is a well-characterized regulatory mechanism in the cytosol, but remains poorly defined in the mitochondrion. In this study, we characterized the use of (32)P-labeling to monitor the turnover of protein phosphorylation in the heart and liver mitochondria matrix. The (32)P labeling technique was compared and contrasted to Phos-tag protein phosphorylation fluorescent stain and 2D isoelectric focusing. Of the 64 proteins identified by MS spectroscopy in the Phos-Tag gels, over 20 proteins were correlated with (32)P labeling. The high sensitivity of (32)P incorporation detected proteins well below the mass spectrometry and even 2D gel protein detection limits. Phosphate-chase experiments revealed both turnover and phosphate associated protein pool size alterations dependent on initial incubation conditions. Extensive weak phosphate/phosphate metabolite interactions were observed using nondisruptive native gels, providing a novel approach to screen for potential allosteric interactions of phosphate metabolites with matrix proteins. We confirmed the phosphate associations in Complexes V and I due to their critical role in oxidative phosphorylation and to validate the 2D methods. These complexes were isolated by immunocapture, after (32)P labeling in the intact mitochondria, and revealed (32)P-incorporation for the alpha, beta, gamma, OSCP, and d subunits in Complex V and the 75, 51, 42, 23, and 13a kDa subunits in Complex I. These results demonstrate that a dynamic and extensive mitochondrial matrix phosphoproteome exists in heart and liver.
View details for DOI 10.1021/pr800913j
View details for Web of Science ID 000266719400008
View details for PubMedID 19351177
Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca(2+)) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent (32)P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca(2+) has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca(2+) signaling network, the effects of a Ca(2+) challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca(2+)-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca(2+) dose-dependent dephosphorylation of MnSOD was associated with an approximately 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca(2+). These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca(2+) modification of mitochondrial function at the level of MnSOD.
View details for DOI 10.1021/bi052475e
View details for Web of Science ID 000235792300008
View details for PubMedID 16489745