High-throughput, big data and complexity in clinical proteomics: an interview with Jasminka Godovac-Zimmermann

Interview with Professor Jasminka Godovac-Zimmermann, PhD by Claire Raison (Commissioning Editor) Professor Jasminka Godovac-Zimmermann is Head of the Proteomics and Molecular Cell Dynamics Group at University College London, UK. Professor Godovac-Zimmermann trained at the Max Planck Institute of Biochemistry, Germany, and specialized in protein chemistry. Her research focuses on proteomics in cancer and systems biology. Here she talks about the clinical impact of her work and her hopes and predictions for how proteomics and diagnostics could work together in future

Moonlighting Proteins and Cardiopathy in the Spatial Response of MCF‐7 Breast Cancer Cells to Tamoxifen

Background: The purpose of this study is to apply quantitative high‐throughput proteomics methods to investigate dynamic aspects of protein changes in nucleocytoplasmic distribution of proteins and of total protein abundance for MCF‐7 cells exposed to tamoxifen (Tam) in order to reveal the agonistic and antagonistic roles of the drug. / Experimental design: The MS‐based global quantitative proteomics with the analysis of fractions enriched in target subcellular locations is applied to measure the changes in total abundance and in the compartmental abundance/distribution between the nucleus and cytoplasm for several thousand proteins differentially expressed in MCF‐7 cells in response to Tam stimulation. / Results: The response of MCF‐7 cells to the Tam treatment shows significant changes in subcellular abundance rather than in their total abundance. The bioinformatics study reveals the relevance of moonlighting proteins and numerous pathways involved in Tam response of MCF‐7 including some of which may explain the agonistic and antagonistic roles of the drug. / Conclusions: The results indicate possible protective role of Tam against cardiovascular diseases as well as its involvement in G‐protein coupled receptors pathways that enhance breast tissue proliferation

Quantitative Proteomics Reveals a "Poised Quiescence" Cellular State after Triggering the DNA Replication Origin Activation Checkpoint

An origin activation checkpoint has recently been discovered in the G1 phase of the mitotic cell cycle, which can be triggered by loss of DNA replication initiation factors such as the Cdc7 kinase. Insufficient levels of Cdc7 activate cell cycle arrest in normal cells, whereas cancer cells appear to lack this checkpoint response, do not arrest, and proceed with an abortive S phase, leading to cell death. The differential response between normal and tumor cells at this checkpoint has led to widespread interest in the development of pharmacological Cdc7 inhibitors as novel anticancer agents. We have used RNAi against Cdc7 in combination with SILAC-based high resolution MS proteomics to investigate the cellular mechanisms underlying the maintenance of the origin activation checkpoint in normal human diploid fibroblasts. Bioinformatics analysis identified clear changes in wide-ranging biological processes including altered cellular energetic flux, moderate stress response, reduced proliferative capacity, and a spatially distributed response across the mitochondria, lysosomes, and the cell surface. These results provide a quantitative overview of the processes involved in maintenance of the arrested state, show that this phenotype involves active rather than passive cellular adaptation, and highlight a diverse set of proteins responsible for cell cycle arrest and ultimately for promotion of cellular survival. We propose that the Cdc7-depleted proteome maintains cellular arrest by initiating a dynamic quiescence-like response and that the complexities of this phenotype will have important implications for the continued development of promising Cdc7-targeted cancer therapies

Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

We have used a subcellular spatial razor approach based on LC–MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function

Quantification of FAM20A in human milk and identification of calcium metabolism proteins

BACKGROUND: FAM20A, a recently discovered protein, is thought to have a fundamental role in inhibiting ectopic calcification. Several studies have demonstrated that variants of FAM20A are causative for the rare autosomal recessive disorder, enamel-renal syndrome (ERS). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. FAM20A is a low-abundant protein that is difficult to detect in biofluids such as blood, saliva, and urine. Thus, we speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Therefore, the primary aim of this research is to describe the processes/methodology taken to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism. METHOD: This study used mass spectrometry-driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions. RESULTS: Shotgun MS/MS driven proteomics identified FAM20A in whole milk, and subsequent analysis using targeted proteomics also successfully quantified FAM20A in all samples. Combination of sample preparation, fractionation, and LC-MS/MS proteomics analysis generated 136 proteins previously undiscovered in human milk; 21 of these appear to be associated with calcium metabolism. CONCLUSION: Using mass spectrometry-driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. Furthermore, we show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk

Optineurin deficiency contributes to impaired cytokine secretion and neutrophil recruitment in bacteria driven colitis.

Crohn's disease (CD) is associated with delayed neutrophil recruitment and bacterial clearance at sites of acute inflammation as a result of impaired secretion of proinflammatory cytokines by macrophages. To investigate the impaired cytokine secretion, we performed transcriptomic analysis in macrophages and identified a subgroup of CD patients with low expression of the autophagy receptor optineurin (OPTN). Here we clarified the role of OPTN deficiency in macrophage cytokine secretion, models of bacteria driven colitis and peritonitis in mice and zebrafish Salmonella infection. OPTN deficient bone-marrow derived macrophages (BMDM) stimulated with heat-killed E. coli secreted less proinflammatory TNF and IL6 cytokines despite similar gene transcription, which normalised with lysosomal and autophagy inhibitors suggesting that TNF is mistrafficked to lysosomes via bafilomycin A dependent pathways in the absence of OPTN. OPTN deficient mice were more susceptible to Citrobacter colitis and E. coli peritonitis with reduced levels of proinflammatory TNF in serum, diminished neutrophil recruitment to sites of acute inflammation and greater mortality. Optn knockdown zebrafish infected with Salmonella also had higher mortality. OPTN plays a role in acute inflammation and neutrophil recruitment, potentially via defective macrophage proinflammatory cytokine secretion, which suggests that diminished OPTN expression in humans may increase the risk of developing CD