Gene Ontology (GO)-Driven Inference of Candidate Proteomic Markers Associated with Muscle Atrophy Conditions.

Skeletal muscle homeostasis is essential for the maintenance of a healthy and active lifestyle. Imbalance in muscle homeostasis has significant consequences such as atrophy, loss of muscle mass, and progressive loss of functions. Aging-related muscle wasting, sarcopenia, and atrophy as a consequence of disease, such as cachexia, reduce the quality of life, increase morbidity and result in an overall poor prognosis. Investigating the muscle proteome related to muscle atrophy diseases has a great potential for diagnostic medicine to identify (i) potential protein biomarkers, and (ii) biological processes and functions common or unique to muscle wasting, cachexia, sarcopenia, and aging alone. We conducted a meta-analysis using gene ontology (GO) analysis of 24 human proteomic studies using tissue samples (skeletal muscle and adipose biopsies) and/or biofluids (serum, plasma, urine). Whilst there were few similarities in protein directionality across studies, biological processes common to conditions were identified. Here we demonstrate that the GO analysis of published human proteomics data can identify processes not revealed by single studies. We recommend the integration of proteomics data from tissue samples and biofluids to yield a comprehensive overview of the human skeletal muscle proteome. This will facilitate the identification of biomarkers and potential pathways of muscle-wasting conditions for use in clinics.

Bile and urine peptide marker profiles: access keys to molecular pathways and biological processes in cholangiocarcinoma.

BACKGROUND: Detection of cholangiocarcinoma (CCA) remains a diagnostic challenge. We established diagnostic peptide biomarkers in bile and urine based on capillary electrophoresis coupled to mass spectrometry (CE-MS) to detect both local and systemic changes during CCA progression. In a prospective cohort study we recently demonstrated that combined bile and urine proteome analysis could further improve diagnostic accuracy of CCA diagnosis in patients with unknown biliary strictures. As a continuation of these investigations, the aim of the present study was to investigate the pathophysiological mechanisms behind the molecular determinants reflected by bile and urine peptide biomarkers. METHODS: Protease mapping and gene ontology cluster analysis were performed for the previously defined CE-MS based biomarkers in bile and urine. For that purpose, bile and urine peptide profiles (from samples both collected at the date of endoscopy) were investigated from a representative cohort of patients with benign (n = 76) or CCA-associated (n = 52) biliary strictures (verified during clinical follow-up). This was supplemented with a literature search for the association of the individual biomarkers included in the proteomic patterns with CCA or cancer progression. RESULTS: For most of the peptide markers, association to CCA has been described in literature. Protease mapping revealed ADAMTS4 activity in cleavage of both bile and urine CCA peptide biomarkers. Furthermore, increased chymase activity in bile points to mast cell activation at the tumor site. Gene ontology cluster analysis indicates cellular response to chemical stimuli and stress response as local and extracellular matrix reorganization by tissue destruction and repair as systemic events. The analysis further supports that the mapped proteases are drivers of local and systemic events. CONCLUSIONS: The study supports connection of the CCA-associated peptide biomarkers to the molecular pathophysiology and indicates an involvement in epithelial-to-mesenchymal transition, generation of cancer-associated fibroblasts and activation of residual immune cells. Proteases, extracellular matrix components, inflammatory cytokines, proangiogenic, growth and vasoactive factors released from the tumor microenvironment are drivers of systemic early events during CCA progression.

The influence of hypoxia on the prostate cancer proteome.

Prostate cancer accounts for around 15% of male deaths in Western Europe and is the second leading cause of cancer death in men after lung cancer. Mounting evidence suggests that prostate cancer deposits exist within a hypoxic environment and this contributes to radio-resistance thus hampering one of the major therapies for this cancer. Recent reports have shown that nitric oxide (NO) donating non-steroidal anti-inflammatory drugs (NSAIDs) reduced tumour hypoxia as well as maintaining a radio-sensitising/therapeutic effect on prostate cancer cells. The aim of this study was to evaluate the impact of hypoxia on the proteome of the prostate and to establish whether NO-NSAID treatment reverted the protein profiles back to their normoxic status. To this end an established hormone insensitive prostate cancer cell line, PC-3, was cultured under hypoxic and normoxic conditions before and following exposure to NO-NSAID in combination with selected other common prostate cancer treatment types. The extracted proteins were analysed by ion mobility-assisted data independent acquisition mass spectrometry (MS), combined with multivariate statistical analyses, to measure hypoxia-induced alterations in the proteome of these cells. The analyses demonstrated that under hypoxic conditions there were well-defined, significantly regulated/differentially expressed proteins primarily involved with structural and binding processes including, for example, TUBB4A, CIRP and PLOD1. Additionally, the exposure of hypoxic cells to NSAID and NO-NSAID agents, resulted in some of these proteins being differentially expressed; for example, both PCNA and HNRNPA1L were down-regulated, corresponding with disruption in the nucleocytoplasmic shuttling process.

MAGE genes in the kidney: identification of MAGED2 as upregulated during kidney injury and in stressed tubular cells.

BACKGROUND: Mutations in Melanoma Antigen-encoding Gene D2 (MAGED2) promote tubular dysfunction, suggesting that MAGE proteins may play a role in kidney pathophysiology. We have characterized the expression and regulation of MAGE genes in normal kidneys and during kidney disease. METHODS: The expression of MAGE genes and their encoded proteins was explored by systems biology multi-omics (kidney transcriptomics and proteomics) in healthy adult murine kidneys and following induction of experimental acute kidney injury (AKI) by a folic acid overdose. Changes in kidney expression during nephrotoxic AKI were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry. Factors regulating gene expression were studied in cultured tubular cells. RESULTS: Five MAGE genes (MAGED1, MAGED2, MAGED3, MAGEH1, MAGEE1) were expressed at the mRNA level in healthy adult mouse kidneys, as assessed by RNA-Seq. Additionally, MAGED2 was significantly upregulated during experimental AKI as assessed by array transcriptomics. Kidney proteomics also identified MAGED2 as upregulated during AKI. The increased kidney expression of MAGED2 mRNA and protein was confirmed by qRT-PCR and western blot, respectively, in murine folic acid- and cisplatin-induced AKI. Immunohistochemistry located MAGED2 to tubular cells in experimental and human kidney injury. Tubular cell stressors [serum deprivation and the inflammatory cytokine tumour necrosis factor-like weak inducer of apoptosis (TWEAK)] upregulated MAGED2 in cultured tubular cells. CONCLUSIONS: MAGED2 is upregulated in tubular cells in experimental and human kidney injury and is increased by stressors in cultured tubular cells. This points to a role of MAGED2 in tubular cell injury during kidney disease that should be dissected by carefully designed functional approaches.

Mitogen-Activated Protein Kinase 14 Promotes AKI.

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry-based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity-deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target.

Acute kidney injury prediction in cardiac surgery patients by a urinary peptide pattern: a case-control validation study.

BACKGROUND: Acute kidney injury (AKI) is a prominent problem in hospitalized patients and associated with increased morbidity and mortality. Clinical medicine is currently hampered by the lack of accurate and early biomarkers for diagnosis of AKI and the evaluation of the severity of the disease. In 2010, we established a multivariate peptide marker pattern consisting of 20 naturally occurring urinary peptides to screen patients for early signs of renal failure. The current study now aims to evaluate if, in a different study population and potentially various AKI causes, AKI can be detected early and accurately by proteome analysis. METHODS: Urine samples from 60 patients who developed AKI after cardiac surgery were analyzed by capillary electrophoresis-mass spectrometry (CE-MS). The obtained peptide profiles were screened by the AKI peptide marker panel for early signs of AKI. Accuracy of the proteomic model in this patient collective was compared to that based on urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) ELISA levels. Sixty patients who did not develop AKI served as negative controls. RESULTS: From the 120 patients, 110 were successfully analyzed by CE-MS (59 with AKI, 51 controls). Application of the AKI panel demonstrated an AUC in receiver operating characteristics (ROC) analysis of 0.81 (95 % confidence interval: 0.72-0.88). Compared to the proteomic model, ROC analysis revealed poorer classification accuracy of NGAL and KIM-1 with the respective AUC values being outside the statistical significant range (0.63 for NGAL and 0.57 for KIM-1). CONCLUSIONS: This study gives further proof for the general applicability of our proteomic multimarker model for early and accurate prediction of AKI irrespective of its underlying disease cause.

Diagnosis and Prediction of CKD Progression by Assessment of Urinary Peptides.

Progressive CKD is generally detected at a late stage by a sustained decline in eGFR and/or the presence of significant albuminuria. With the aim of early and improved risk stratification of patients with CKD, we studied urinary peptides in a large cross-sectional multicenter cohort of 1990 individuals, including 522 with follow-up data, using proteome analysis. We validated that a previously established multipeptide urinary biomarker classifier performed significantly better in detecting and predicting progression of CKD than the current clinical standard, urinary albumin. The classifier was also more sensitive for identifying patients with rapidly progressing CKD. Compared with the combination of baseline eGFR and albuminuria (area under the curve [AUC]=0.758), the addition of the multipeptide biomarker classifier significantly improved CKD risk prediction (AUC=0.831) as assessed by the net reclassification index (0.303±-0.065; P<0.001) and integrated discrimination improvement (0.058±0.014; P<0.001). Correlation of individual urinary peptides with CKD stage and progression showed that the peptides that associated with CKD, irrespective of CKD stage or CKD progression, were either fragments of the major circulating proteins, suggesting failure of the glomerular filtration barrier sieving properties, or different collagen fragments, suggesting accumulation of intrarenal extracellular matrix. Furthermore, protein fragments associated with progression of CKD originated mostly from proteins related to inflammation and tissue repair. Results of this study suggest that urinary proteome analysis might significantly improve the current state of the art of CKD detection and outcome prediction and that identification of the urinary peptides allows insight into various ongoing pathophysiologic processes in CKD.

Cerebrospinal fluid prohormone processing and neuropeptides stimulating feed intake of dairy cows during early lactation.

After parturition, feed intake of dairy cows increases within the first weeks of lactation, but the molecular mechanisms stimulating or delaying the slope of increase are poorly understood. Some of the molecules controlling feed intake are neuropeptides that are synthesized as propeptides and subsequently processed before they bind to specific receptors in feeding centers of the brain. Cerebrospinal fluid surrounds most of the feed intake regulatory centers and contains numerous neuropeptides. In the present study, we used a proteomic approach to analyze the neuropeptide concentrations in cerebrospinal fluid taken from dairy cows between day -18 and -10, and between day +10 and +20 relative to parturition. We found 13 proteins which were only present in samples taken before parturition, 13 proteins which were only present in samples taken after parturition, and 25 proteins which were commonly present, before and after parturition. Among them, differences in pro-neuropeptide Y, proenkephalin-A, neuroendocrine convertase-2, neurosecretory protein VGF, chromogranin-A, and secretogranin-1 and -3 concentrations relative to parturition highlight propeptides and prohormone processings involved in the control of feed intake and energy homeostasis. Scaffold analysis further emphasized an increased tone of endogenous opioids associated with the postparturient increase of feed intake.

New insights in molecular mechanisms involved in chronic kidney disease using high-resolution plasma proteome analysis.

BACKGROUND: The reduced glomerular filtration rate in the advanced stages of chronic kidney disease (CKD) leads to plasma accumulation of uraemic retention solutes including proteins. It has been hypothesized that these changes may, at least in part, be responsible for CKD-associated morbidity and mortality. However, most studies focused on the role of individual proteins, while a holistic, large-scale, integrative approach may generate significant additional insight. METHODS: In a discovery study, we analysed the plasma proteome of patients with stage 2-3 CKD (n = 14) and stage 5 CKD with haemodialysis (HD) (n = 15), using high-resolution LC-MS/MS analysis. Selected results were validated in a cohort of 40 patients with different CKD stages with or without HD, using ELISA. RESULTS: Of a total of 2054 detected proteins, 127 displayed lower, while 206 displayed higher abundance in the plasma of patients on HD. Molecular pathway analysis confirmed the modification of known processes involved in CKD complications, including decreased haemostasis and increased inflammation, complement activation and vascular damage. In addition, we identified the plasma increase during CKD progression of lysozyme C and leucine-rich alpha-2 glycoprotein, two proteins related to vascular damage and heart failure. High level of leucine-rich alpha-2 glycoprotein was associated with higher mortality in stage 5 CKD patients on HD. CONCLUSIONS: This study provides for the first time a comprehensive assessment of CKD plasma proteome, contributing to new knowledge and potential markers of CKD. These results will serve as a basis for future studies investigating the relevance of these molecules in CKD associated morbidity and mortality.

Increased sputum endotoxin levels are associated with an impaired lung function response to oral steroids in asthmatic patients.

BACKGROUND: Airway endotoxin might contribute to corticosteroid insensitivity in asthmatic patients. OBJECTIVE: Because cigarette smoke contains endotoxin, we tested the hypothesis that sputum endotoxin concentrations are increased in cigarette smokers and that endotoxin concentrations are associated with corticosteroid insensitivity in asthmatic patients. METHODS: Sixty-nine asthmatic patients (never smokers, smokers, and exsmokers) and 20 healthy subjects (never smokers and smokers) were recruited. Fifty-three asthmatic patients received a 2-week course of oral dexamethasone. Serum and induced sputum endotoxin and cytokine concentrations were quantified by using an enzyme immunoassay. RESULTS: Median (interquartile range [IQR]) sputum endotoxin concentration were not significantly different between asthmatic never smokers (184 endotoxin units [EU]/mL; IQR, 91-310 EU/mL), exsmokers (123 EU/mL; IQR, 39-207 EU/mL), and smokers (177 EU/mL; IQR, 41-772 EU/mL; P = .703) and healthy subjects (164 EU/mL; IQR, 106-373 EU/mL). The lung function response to oral corticosteroids decreased with increasing sputum endotoxin concentrations in the never smokers (linear regression α = .05, Spearman r = -0.503, P = .009) but not in smokers (α = .587, r = -0.282, P = .257), as confirmed by using multiple regression analysis. Asthmatic smokers had higher concentrations of serum endotoxin than asthmatic nonsmokers (0.25 EU/mL [IQR, 0.09-0.39 EU/mL] vs 0.08 EU/mL [IQR, 0.05-0.19 EU/mL], P = .042) unrelated to steroid insensitivity or serum cytokine concentrations. In the asthmatic group sputum endotoxin concentrations correlated with sputum IL-1 receptor antagonist concentrations (r = 0.510, P < .001), and serum endotoxin concentrations significantly correlated with sputum IL-6, IL-8, and chemokine motif ligand 2 concentrations. CONCLUSION: Asthmatic smokers have similar sputum endotoxin concentrations compared with those of asthmatic never smokers. The association between higher sputum endotoxin levels and an impaired lung function response to oral corticosteroids, particularly in asthmatic never smokers, suggests that airway endotoxin might contribute to corticosteroid insensitivity in asthmatic patients.

Improving peptide relative quantification in MALDI-TOF MS for biomarker assessment.

Proteomic profiling by MALDI-TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI-TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI-TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI-TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).

Performance benchmarking microplate-immunoassays for quantifying target-specific cysteine oxidation reveals their potential for understanding redox-regulation and oxidative stress.

The antibody-linked oxi-state assay (ALISA) for quantifying target-specific cysteine oxidation can benefit specialist and non-specialist users. Specialists can benefit from time-efficient analysis and high-throughput target and/or sample n-plex capacities. The simple and accessible "off-the-shelf" nature of ALISA brings the benefits of oxidative damage assays to non-specialists studying redox-regulation. Until performance benchmarking establishes confidence in the "unseen" microplate results, ALISA is unlikely to be widely adopted. Here, we implemented pre-set pass/fail criteria to benchmark ALISA by robustly evaluating immunoassay performance in diverse biological contexts. ELISA-mode ALISA assays were accurate, reliable, and sensitive. For example, the average inter-assay CV for detecting 20%- and 40%-oxidised PRDX2 or GAPDH standards was 4.6% (range: 3.6-7.4%). ALISA displayed target-specificity. Immunodepleting the target decreased the signal by ∼75%. Single-antibody formatted ALISA failed to quantify the matrix-facing alpha subunit of the mitochondrial ATP synthase. However, RedoxiFluor quantified the alpha subunit displaying exceptional performance in the single-antibody format. ALISA discovered that (1) monocyte-to-macrophage differentiation amplified PRDX2-specific cysteine oxidation in THP-1 cells and (2) exercise increased GAPDH-specific cysteine oxidation in human erythrocytes. The "unseen" microplate data were "seen-to-be-believed" via orthogonal visually displayed immunoassays like the dimer method. Finally, we established target (n = 3) and sample (n = 100) n-plex capacities in ∼4 h with 50-70 min hands-on time. Our work showcases the potential of ALISA to advance our understanding of redox-regulation and oxidative stress.

Integrative OMICS Data-Driven Procedure Using a Derivatized Meta-Analysis Approach.

The wealth of high-throughput data has opened up new opportunities to analyze and describe biological processes at higher resolution, ultimately leading to a significant acceleration of scientific output using high-throughput data from the different omics layers and the generation of databases to store and report raw datasets. The great variability among the techniques and the heterogeneous methodologies used to produce this data have placed meta-analysis methods as one of the approaches of choice to correlate the resultant large-scale datasets from different research groups. Through multi-study meta-analyses, it is possible to generate results with greater statistical power compared to individual analyses. Gene signatures, biomarkers and pathways that provide new insights of a phenotype of interest have been identified by the analysis of large-scale datasets in several fields of science. However, despite all the efforts, a standardized regulation to report large-scale data and to identify the molecular targets and signaling networks is still lacking. Integrative analyses have also been introduced as complementation and augmentation for meta-analysis methodologies to generate novel hypotheses. Currently, there is no universal method established and the different methods available follow different purposes. Herein we describe a new unifying, scalable and straightforward methodology to meta-analyze different omics outputs, but also to integrate the significant outcomes into novel pathways describing biological processes of interest. The significance of using proper molecular identifiers is highlighted as well as the potential to further correlate molecules from different regulatory levels. To show the methodology's potential, a set of transcriptomic datasets are meta-analyzed as an example.

ORA , FCS , and PT Strategies in Functional Enrichment Analysis.

Downstream analysis of OMICS data requires interpretation of many molecular components considering current biological knowledge. Most tools used at present for functional enrichment analysis workflows applied to the field of proteomics are either borrowed or have been modified from genomics workflows to accommodate proteomics data. While the field of proteomics data analytics is evolving, as is the case for molecular annotation coverage, one can expect the rise of enhanced databases with less redundant ontologies spanning many elements of the tree of life. The methodology described here shows in practical steps how to perform overrepresentation analysis, functional class scoring, and pathway-topology analysis using a preexisting neurological dataset of proteomic data.

Integrative Analysis of Incongruous Cancer Genomics and Proteomics Datasets.

Cancer is a complex disease characterized by molecular heterogeneity and the involvement of several cellular mechanisms throughout its evolution and pathogenesis. Despite the great efforts made to untangle these mechanisms, cancer pathophysiology remains far from clear. So far, panels of biomarkers have been reported from high-throughput data generated through different platforms. These biomarkers are primarily focused on one type of coding molecules such as transcripts or proteins, mainly due to the apparent heterogeneity of output data resulting from the use of various techniques specific to the molecular type. Hence, there is a major need to understand how these molecules interact and complement each other to be able to explain the deregulated processes involved. The breadth of large-scale data availability as well as the lack of in-depth analysis of publicly available data has raised concerns and enabled opportunities for new strategies to analyze "Big data" more comprehensively. Here, a new protocol to perform integrative analysis based on a systems biology approach is described. The foundation of the approach relies on groups of datasets from published studies compared within the original described groups and organized in a designated format to allow the integration and cross-comparison among different studies and different platforms. This approach follows an unbiased hypothesis-free methodology that will facilitate the identification of commonalities among different data-set sources, and ultimately map and characterize specific molecular pathways using significantly deregulated molecules. This in turn will generate novel insights about the mechanisms deregulated in complex diseases such as cancer.

Selective chemical intervention in the proteome of Caenorhabditis elegans.

We present the first study of protein regulation by ligands in Caenorhabditis elegans. The ligands were peptidyl-prolyl isomerase inhibitors of cyclophilins. Up-regulation is observed for several heat shock proteins and one ligand in particular caused a greater than 2-fold enhancement of cyclophilin CYN-5. Additionally, several metabolic enzymes display elevated levels. This approach, using label-free relative quantification, provides an extremely attractive way of measuring the effect of ligands on an entire proteome, with minimal sample pretreatment, which could be applicable to large-scale studies. In this initial study, which compares the effect of three ligands, 54 unique proteins have been identified that are up- (51) or down- (3) regulated in the presence of a given ligand. A total of 431 C. elegans proteins were identified. Our methodology provides an intriguing new direction for in vivo screening of the effects of novel and untested ligands at the whole organism level.

Immunological Techniques to Assess Protein Thiol Redox State: Opportunities, Challenges and Solutions.

To understand oxidative stress, antioxidant defense, and redox signaling in health and disease it is essential to assess protein thiol redox state. Protein thiol redox state is seldom assessed immunologically because of the inability to distinguish reduced and reversibly oxidized thiols by Western blotting. An underappreciated opportunity exists to use Click PEGylation to realize the transformative power of simple, time and cost-efficient immunological techniques. Click PEGylation harnesses selective, bio-orthogonal Click chemistry to separate reduced and reversibly oxidized thiols by selectively ligating a low molecular weight polyethylene glycol moiety to the redox state of interest. The resultant ability to disambiguate reduced and reversibly oxidized species by Western blotting enables Click PEGylation to assess protein thiol redox state. In the present review, to enable investigators to effectively harness immunological techniques to assess protein thiol redox state we critique the chemistry, promise and challenges of Click PEGylation.

Reversible Thiol Oxidation Inhibits the Mitochondrial ATP Synthase in Xenopus Laevis Oocytes.

Oocytes are postulated to repress the proton pumps (e.g., complex IV) and ATP synthase to safeguard mitochondrial DNA homoplasmy by curtailing superoxide production. Whether the ATP synthase is inhibited is, however, unknown. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min-1/mg-1) in testes compared to oocytes in Xenopus laevis (X. laevis). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. If a protein thiol inhibits the ATP synthase, then constituent subunits must be reversibly oxidised. Catalyst-free trans-cyclooctene 6-methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting reveals several subunits in F1 (e.g., ATP-α-F1) and Fo (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-α-F1 oxidation at two evolutionary conserved cysteine residues (C244 and C294) in oocytes. TCO-Tz Click PEGylation reveals ~20% of the total thiols in the ATP synthase are substantially oxidised. Chemically reversing thiol oxidation significantly increased oligomycin sensitive ATP synthase activity from ~12 to 100 nmol/min-1/mg-1 in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in X. laevis oocytes.

Proteomic strategies to unravel age-related redox signalling defects in skeletal muscle.

Increased oxidative damage and disrupted redox signalling are consistently associated with age-related loss of skeletal muscle mass and function. Redox signalling can directly regulate biogenesis and degradation pathways and indirectly via activation of key transcription factors. Contracting skeletal muscle fibres endogenously generate free radicals (e.g. superoxide) and non-radical derivatives (e.g. hydrogen peroxide). Exercise induced redox signalling can promote beneficial adaptive responses that are disrupted by age-related redox changes. Identifying and quantifying the redox signalling pathways responsible for successful adaptation to exercise makes skeletal muscle an attractive physiological model for redox proteomic approaches. Site specific identification of the redox modification and quantification of site occupancy in the context of protein abundance remains a crucial concept for redox proteomics approaches. Notwithstanding, the technical limitations associated with skeletal muscle for proteomic analysis, we discuss current approaches for the identification and quantification of transient and stable redox modifications that have been employed to date in ageing research. We also discuss recent developments in proteomic approaches in skeletal muscle and potential implications and opportunities for investigating disrupted redox signalling in skeletal muscle ageing.

Acute stress alters the rates of degradation of cardiac muscle proteins.

Stressful experiences can have detrimental effects on many aspects of health and wellbeing. The zebrafish (Danio rerio) is a widely used model for stress research and a stress phenotype can be induced by manipulating the environmental conditions and social interactions. In this study we have combined a zebrafish stress model with the measurement of degradation rates of soluble cardiac muscle proteins. The results showed that the greater the stress response in the zebrafish the lower the level of overall protein degradation. On comparing the rates of degradation for individual proteins it was found that four main pathways were altered in response to stress conditions with decreased degradation for proteins involved in glucose metabolism, gluconeogenesis, the ubiquitin-proteasome system (UPS) and peroxisomal proliferator-activated receptor (PPAR) signalling pathways. Taken together, these data indicate that under stress conditions zebrafish preserve cardiac muscle proteins required for the 'fight or flight' response together with proteins that play a role in stress mitigation. SIGNIFICANCE: This study is the first to investigate the impact of stressful experiences on the dynamics of protein turnover in cardiac muscle. Using an established zebrafish model of human stress it has been possible to map key pathways at the protein level. The results show that the rates of degradation of cardiac proteins involved in glucose metabolism, UPS activity, hypoxia and PPAR signalling are decreased in stressed zebrafish. These findings indicate that proteins involved in the 'fight or flight' response to stress are conserved by the heart together with proteins that play a role in stress mitigation. This work provides the basis for more detailed investigations aimed at understanding the molecular effects of stress, which has implications for human health and disease.