Human Dopaminergic Neurons Lacking PINK1 Exhibit Disrupted Dopamine Metabolism Related to Vitamin B6 Co-Factors.

PINK1 loss-of-function mutations cause early onset Parkinson disease. PINK1-Parkin mediated mitophagy has been well studied, but the relevance of the endogenous process in the brain is debated. Here, the absence of PINK1 in human dopaminergic neurons inhibits ionophore-induced mitophagy and reduces mitochondrial membrane potential. Compensatory, mitochondrial renewal maintains mitochondrial morphology and protects the respiratory chain. This is paralleled by metabolic changes, including inhibition of the TCA cycle enzyme mAconitase, accumulation of NAD+, and metabolite depletion. Loss of PINK1 disrupts dopamine metabolism by critically affecting its synthesis and uptake. The mechanism involves steering of key amino acids toward energy production rather than neurotransmitter metabolism and involves cofactors related to the vitamin B6 salvage pathway identified using unbiased multi-omics approaches. We propose that reduction of mitochondrial membrane potential that cannot be controlled by PINK1 signaling initiates metabolic compensation that has neurometabolic consequences relevant to Parkinson disease.

Absolute Quantification of Endogenous Ras Isoform Abundance.

Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data.

Differential reprogramming of isogenic colorectal cancer cells by distinct activating KRAS mutations.

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼ 16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.

The role of palmitoylation in regulating Ras localization and function.

Ras GTPases are important regulators of pathways controlling proliferation, differentiation and transformation. Three ubiquitously expressed almost identical Ras genes are not functionally redundant; this has been attributed to their distinctive trafficking and localization profiles. A palmitoylation cycle controls the correct compartmentalization of H-Ras and N-Ras. We review recent data that reveal how this cycle can be regulated by membrane organization to influence the spatiotemporal signalling of Ras.

PROTEINCHALLENGE: crowd sourcing in proteomics analysis and software development.

In large-scale proteomics studies there is a temptation, after months of experimental work, to plug resulting data into a convenient-if poorly implemented-set of tools, which may neither do the data justice nor help answer the scientific question. In this paper we have captured key concerns, including arguments for community-wide open source software development and "big data" compatible solutions for the future. For the meantime, we have laid out ten top tips for data processing. With these at hand, a first large-scale proteomics analysis hopefully becomes less daunting to navigate. However there is clearly a real need for robust tools, standard operating procedures and general acceptance of best practises. Thus we submit to the proteomics community a call for a community-wide open set of proteomics analysis challenges--PROTEINCHALLENGE--that directly target and compare data analysis workflows, with the aim of setting a community-driven gold standard for data handling, reporting and sharing.

Miniaturisation of a high throughput screening assay comparing air displacement and capillary-based nanolitre transfer technologies.

The miniaturisation of high-throughput screening (HTS) assays has been a widely debated and researched strategy with the aims of reducing screening costs and increasing speed while not compromising data quality. In this context, liquid handling technologies continue to be improved. A new and promising development is the emergence of nanolitre dispensers, which are capable of direct compound transfer to assay microplates. In this study, we investigated the miniaturisation of a HTS kinase assay and compared the real life performance of current state-of-the-art air displacement transfer technology (MiniTrak V System) and a capillary based nanolitre dispenser (CyBi-HummingWell). The robustness and effectiveness of the miniaturised assay formats were compared by testing staurosporine to generate dose-response curves and 340 previously identified active compounds. Using the MiniTrak device, assay miniaturisation was achieved from 18 microL to 6 microL in 384-well and 1536-well plate formats. Utilising the nanolitre dispenser, miniaturisation was performed down to 5 microL in 1536-well plates. The Z' factors obtained for each assay format were consistently above 0.5. The data presented here describe the reproducibility of the results obtained with the two transfer technologies and highlight possible issues for hit identification.