Harvey E. Johnston

Whole Serum 3D LC-nESI-FTMS Quantitative Proteomics Reveals Sexual Dimorphism in the Milieu Intérieur of Overweight and Obese Adults

Linking gender-specific differences to the molecular etiology of obesity has been largely based on genomic and transcriptomic evidence lacking endophenotypic insight and is not applicable to the extracellular fluid compartments, or the milieu intérieur, of the human body. To address this need, this study profiled the whole serum proteomes of age-matched nondiabetic overweight and obese females (n = 28) and males (n = 31) using a multiplex design with pooled biological and technical replicates. To bypass basic limitations of immunodepletion-based strategies, subproteome enrichment by size-exclusion chromatography (SuPrE-SEC) followed by iTRAQ 2D-LC-nESI-FTMS analysis was used. The study resulted in the reproducible analysis of 2472 proteins (peptide FDR < 5%, q < 0.05). A total of 248 proteins exhibited significant modulation between men and women (p < 0.05) that mapped to pathways associated with β-estradiol, lipid and prostanoid metabolism, vitamin D function, immunity/inflammation, and the complement and coagulation cascades. This novel endophenotypic signature of gender-specific differences in whole serum confirmed and expanded the results of previous physiologic and pharmacologic studies exploring sexual dimorphism at the genomic and transcriptomic level in tissues and cells. Conclusively, the multifactorial and pleiotropic nature of human obesity exhibits sexual dimorphism in the circulating proteome of importance to clinical study design.

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-β treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.

BDNF Stimulation of Protein Synthesis in Cortical Neurons Requires the MAP Kinase-Interacting Kinase MNK1

Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m7GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study

Background:Prostate cancer (PCa) is the most common male cancer in the United Kingdom and we aimed to identify clinically relevant biomarkers corresponding to stage progression of the disease.Methods:We used enhanced proteomic profiling of PCa progression using iTRAQ 3D LC mass spectrometry on high-quality serum samples to identify biomarkers of PCa.Results:We identified >1000 proteins. Following specific inclusion/exclusion criteria we targeted seven proteins of which two were validated by ELISA and six potentially interacted forming an ‘interactome’ with only a single protein linking each marker. This network also includes accepted cancer markers, such as TNF, STAT3, NF-κB and IL6.Conclusions:Our linked and interrelated biomarker network highlights the potential utility of six of our seven markers as a panel for diagnosing PCa and, critically, in determining the stage of the disease. Our validation analysis of the MS-identified proteins found that SAA alongside KLK3 may improve categorisation of PCa than by KLK3 alone, and that TSR1, although not significant in this model, might also be a clinically relevant biomarker.

Are you also what your mother eats? Distinct proteomic portrait as a result of maternal high-fat diet in the cerebral cortex of the adult mouse

Epidemiological studies suggest an association between maternal obesity and adverse neurodevelopmental outcomes in offspring. Our aim was to compare the global proteomic portrait in the cerebral cortex between mice born to mothers on a high-fat or control diet who themselves were fed a high-fat or control diet. Male mice born to dams fed a control (C) or high-fat (H) diet 4 weeks before conception and during gestation, and lactation were assigned to either C or H diet at weaning. Mice were killed at 19 weeks and their cerebral cortices were analysed using a two-dimensional liquid chromatography-mass spectrometry methodology. In total, 6 695 proteins were identified (q<0.01), 10% of which were modulated in at least one of the groups relative to controls. In silico analysis revealed that mice clustered based on the diet of the mother and not their own diet and that maternal high-fat diet was significantly associated with response to hypoxia/oxidative stress and apoptosis in the cerebral cortex of the adult offspring. Maternal high-fat diet resulted in distinct endophenotypic changes of the adult offspring cerebral cortex independent of its current diet. The identified proteins could represent novel therapeutic targets for the prevention of neuropathological features resulting from maternal obesity.

Integrated Cellular and Plasma Proteomics of Contrasting B-cell Cancers Reveals Common, Unique and Systemic Signatures

Approximately 800,000 leukemia and lymphoma cases are diagnosed worldwide each year. Burkitts lymphoma (BL) and chronic lymphocytic leukemia (CLL) are examples of contrasting B-cell cancers; BL is a highly aggressive lymphoid tumor, frequently affecting children, whereas CLL typically presents as an indolent, slow-progressing leukemia affecting the elderly. The B-cell-specific overexpression of the myc and TCL1 oncogenes in mice induce spontaneous malignancies modeling BL and CLL, respectively. Quantitative mass spectrometry proteomics and isobaric labeling were employed to examine the biology underpinning contrasting Eμ-myc and Eμ-TCL1 B-cell tumors. Additionally, the plasma proteome was evaluated using subproteome enrichment to interrogate biomarker emergence and the systemic effects of tumor burden. Over 10,000 proteins were identified (q<0.01) of which 8270 cellular and 2095 plasma proteins were quantitatively profiled. A common B-cell tumor signature of 695 overexpressed proteins highlighted ribosome biogenesis, cell-cycle promotion and chromosome segregation. Eμ-myc tumors overexpressed several methylating enzymes and underexpressed many cytoskeletal components. Eμ-TCL1 tumors specifically overexpressed ER stress response proteins and signaling components in addition to both subunits of the interleukin-5 (IL5) receptor. IL5 treatment promoted Eμ-TCL1 tumor proliferation, suggesting an amplification of IL5-induced AKT signaling by TCL1. Tumor plasma contained a substantial tumor lysis signature, most prominent in Eμ-myc plasma, whereas Eμ-TCL1 plasma contained signatures of immune-response, inflammation and microenvironment interactions, with putative biomarkers in early-stage cancer. These findings provide a detailed characterization of contrasting B-cell tumor models, identifying common and specific tumor mechanisms. Integrated plasma proteomics allowed the dissection of a systemic response and a tumor lysis signature present in early- and late-stage cancers, respectively. Overall, this study suggests common B-cell cancer signatures exist and illustrates the potential of the further evaluation of B-cell cancer subtypes by integrative proteomics.

Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells.