SUMMARY Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson’s disease proteins, the Ser/Thr-kinase PINK1 and ubiquitin-ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.

Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.

Medulloblastomas are the most common malignant brain tumors in children. Several large-scale genomic studies have detailed their heterogeneity, defining multiple subtypes with unique molecular profiles and clinical behavior. Increased expression of the miR-183∼96∼182 cluster of microRNAs has been noted in several subgroups, including the most clinically aggressive subgroup associated with genetic amplification of MYC. To understand the contribution of miR-183∼96∼182 to the pathogenesis of this aggressive subtype of medulloblastoma, we analyzed global gene expression and proteomic changes that occur upon modulation of miRNAs in this cluster individually and as a group in MYC-amplified medulloblastoma cells. Knockdown of the full miR-183∼96∼182 cluster results in enrichment of genes associated with apoptosis and dysregulation of the PI3K/AKT/mTOR signaling axis. Conversely, there is a relative enrichment of pathways associated with migration, metastasis and epithelial to mesenchymal transition, as well as pathways associated with dysfunction of DNA repair in cells with preserved miR-183 cluster expression. Immunocytochemistry and FACS analysis confirm induction of apoptosis upon knockdown of the miR-183 cluster. Importantly, cell-based migration and invasion assays verify the positive regulation of cell motility/migration by the miR-183 cluster, which is largely mediated by miR-182. We show that the effects on cell migration induced by the miR-183 cluster are coupled to the PI3K/AKT/mTOR pathway through differential regulation of AKT1 and AKT2 isoforms. Furthermore, we show that rapamycin inhibits cell motility/migration in medulloblastoma cells and phenocopies miR-183 cluster knockdown. Thus, the miR-183 cluster regulates multiple biological programs that converge to support the maintenance and metastatic potential of medulloblastoma.

Mitochondrial crista structure partitions vital cellular reactions and is precisely regulated by diverse cellular signals. Here, we show that, in Drosophila, mitochondrial cristae undergo dynamic remodeling among distinct subcellular regions and the Parkinson's disease (PD)-linked Ser/Thr kinase PINK1 participates in their regulation. Mitochondria increase crista junctions and numbers in selective subcellular areas, and this remodeling requires PINK1 to phosphorylate the inner mitochondrial membrane protein MIC60/mitofilin, which stabilizes MIC60 oligomerization. Expression of MIC60 restores crista structure and ATP levels of PINK1-null flies and remarkably rescues their behavioral defects and dopaminergic neurodegeneration. In an extension to human relevance, we discover that the PINK1-MIC60 pathway is conserved in human neurons, and expression of several MIC60 coding variants in the mitochondrial targeting sequence found in PD patients in Drosophila impairs crista junction formation and causes locomotion deficits. These findings highlight the importance of maintenance and plasticity of crista junctions to cellular homeostasis in vivo.