Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control.

Objectives The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. Design and methods The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. Results In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67–0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. Conclusions We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Abstract. Molecular evolutionary analysis of the glyceraldehyde 3-phosphate dehydrogenase (GapC) gene family was conducted in the plant genus Amsinckia (Boraginaceae), a group that exhibits marked variation in the mating system. GapC genes in this group differ from those of Arabidopsis thaliana in terms of both intron size and number. Phylogenetic and Southern hybridization analyses suggest the presence of multiple GapC loci, each defined by a set of base substitutions that are in strong linkage disequilibrium. One species of Amsinckia, A. spectabilis, was studied in some detail. This species consists of selfing (A. s. spectabilis) and outcrossing (A. s. microcarpa) varieties. Two selfing populations and one outcrossing population sample were analyzed in detail for variation at one of the members of this gene family, GapC3. A reduction in number of GapC3 haplotypes and level of genetic diversity was observed in the selfing populations of A. spectabilis. GapC3 in the outcrossing population (but not the two selfing populations) exhibited a significant departure from neutrality in the direction of an excess of singletons. These results are discussed in the context of forces acting on sequence evolution in populations with different mating systems.

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