Affinity Purification-Mass Spectrometry and Single Fiber Physiology/Proteomics Reveals Mechanistic Insights of C18ORF25.

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, which revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localized to the nucleus and the contractile apparatus in the skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled a detailed assessment of various steps in the excitation-contraction pathway including SR calcium handling and force generation, followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein/phenotype associations and 36 fiber-type specific differences, following loss of C18Orf25. Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

Human proximal tubular epithelial cell-derived small extracellular vesicles mediate synchronized tubular ferroptosis in hypoxic kidney injury.

Hypoxia is the key pathobiological trigger of tubular oxidative stress and cell death that drives the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). The mitochondrial-rich proximal tubular epithelial cells (PTEC) are uniquely sensitive to hypoxia and thus, are pivotal in propagating the sustained tubular loss of AKI-to-CKD transition. Here, we examined the role of PTEC-derived small extracellular vesicles (sEV) in propagating the 'wave of tubular death'. Ex vivo patient-derived PTEC were cultured under normoxia (21 % O2) and hypoxia (1 % O2) on Transwell inserts for isolation and analysis of sEV secreted from apical versus basolateral PTEC surfaces. Increased numbers of sEV were secreted from the apical surface of hypoxic PTEC compared with normoxic PTEC. No differences in basolateral sEV numbers were observed between culture conditions. Biological pathway analysis of hypoxic-apical sEV cargo identified distinct miRNAs linked with cellular injury pathways. In functional assays, hypoxic-apical sEV selectively induced ferroptotic cell death (↓glutathione peroxidase-4, ↑lipid peroxidation) in autologous PTEC compared with normoxic-apical sEV. The addition of ferroptosis inhibitors, ferrostatin-1 and baicalein, attenuated PTEC ferroptosis. RNAse A pretreatment of hypoxic-apical sEV also abrogated PTEC ferroptosis, demonstrating a role for sEV RNA in ferroptotic 'wave of death' signalling. In line with these in vitro findings, in situ immunolabelling of diagnostic kidney biopsies from AKI patients with clinical progression to CKD (AKI-to-CKD transition) showed evidence of ferroptosis propagation (increased numbers of ACSL4+ PTEC), while urine-derived sEV (usEV) from these 'AKI-to-CKD transition' patients triggered PTEC ferroptosis (↑lipid peroxidation) in functional studies. Our data establish PTEC-derived apical sEV and their intravesicular RNA as mediators of tubular lipid peroxidation and ferroptosis in hypoxic kidney injury. This concept of how tubular pathology is propagated from the initiating insult into a 'wave of death' provides novel therapeutic check-points for targeting AKI-to-CKD transition.

Discovery and validation of serum glycoprotein biomarkers for high grade serous ovarian cancer.

PURPOSE: This study aimed to identify serum glycoprotein biomarkers for early detection of high-grade serous ovarian cancer (HGSOC), the most common and aggressive histotype of ovarian cancer. EXPERIMENTAL DESIGN: The glycoproteomics pipeline lectin magnetic bead array (LeMBA)-mass spectrometry (MS) was used in age-matched case-control serum samples. Clinical samples collected at diagnosis were divided into discovery (n = 30) and validation (n = 98) sets. We also analysed a set of preclinical sera (n = 30) collected prior to HGSOC diagnosis in the UK Collaborative Trial of Ovarian Cancer Screening. RESULTS: A 7-lectin LeMBA-MS/MS discovery screen shortlisted 59 candidate proteins and three lectins. Validation analysis using 3-lectin LeMBA-multiple reaction monitoring (MRM) confirmed elevated A1AT, AACT, CO9, HPT and ITIH3 and reduced A2MG, ALS, IBP3 and PON1 glycoforms in HGSOC. The best performing multimarker signature had 87.7% area under the receiver operating curve, 90.7% specificity and 70.4% sensitivity for distinguishing HGSOC from benign and healthy groups. In the preclinical set, CO9, ITIH3 and A2MG glycoforms were altered in samples collected 11.1 ± 5.1 months prior to HGSOC diagnosis, suggesting potential for early detection. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings provide evidence of candidate early HGSOC serum glycoprotein biomarkers, laying the foundation for further study in larger cohorts.

Semi-Automated Lectin Magnetic Bead Array (LeMBA) for Translational Serum Glycoprotein Biomarker Discovery and Validation.

Aberrant protein glycosylation is a characteristic of diverse diseases which has been explored as biomarkers. To support translational serum glycoprotein biomarker discovery and validation, we developed a semi-automated workflow using individual lectin-coupled magnetic beads to conduct lectin pulldowns in a high-throughput format. Lectins are naturally occurring glycoprotein binding proteins widely used in glycobiology. While lectin-affinity isolation has been coupled to mass spectrometry-based proteomics, the lectin magnetic bead array (LeMBA) platform allows technically robust screening and measurement of clinical cohorts. This chapter describes detailed lectin-magnetic bead coupling, serum denaturation, lectin magnetic bead pulldown, and on-bead trypsin digest. The resulting tryptic peptides are analyzed by untargeted or targeted liquid chromatography-mass spectrometry (LC-MS), for biomarker discovery, or qualification/validation, respectively. LeMBA-MS generates quantitative data for glycoforms based on lectin affinity of the glycoprotein coupled with MS measurement of one or more prototypic peptides and has successfully been used to discover and validate novel serum cancer glycoprotein biomarkers. This chapter includes detailed protocols for two different liquid handlers, along with recommendations on quality control measures for clinical biomarker studies.

Western Diet Induced Remodelling of the Tongue Proteome.

The tongue is a heavily innervated and vascularized striated muscle that plays an important role in vocalization, swallowing and digestion. The surface of the tongue is lined with papillae which contain gustatory cells expressing various taste receptors. There is growing evidence to suggest that our perceptions of taste and food preference are remodelled following chronic consumption of Western diets rich in carbohydrate and fats. Our sensitivity to taste and also to metabolising Western diets may be a key factor in the rising prevalence of obesity; however, a systems-wide analysis of the tongue is lacking. Here, we defined the proteomic landscape of the mouse tongue and quantified changes following chronic consumption of a chow or Western diet enriched in lipid, fructose and cholesterol for 7 months. We observed a dramatic remodelling of the tongue proteome including proteins that regulate fatty acid and mitochondrial metabolism. Furthermore, the expressions of several receptors, metabolic enzymes and hormones were differentially regulated, and are likely to provide novel therapeutic targets to alter taste perception and food preference to combat obesity.

Molecular and functional profiling of apical versus basolateral small extracellular vesicles derived from primary human proximal tubular epithelial cells under inflammatory conditions.

Proximal tubular epithelial cells (PTEC) are central players in inflammatory kidney diseases. However, the complex signalling mechanism/s via which polarized PTEC mediate disease progression are poorly understood. Small extracellular vesicles (sEV), including exosomes, are recognized as fundamental components of cellular communication and signalling courtesy of their molecular cargo (lipids, microRNA, proteins). In this study, we examined the molecular content and function of sEV secreted from the apical versus basolateral surfaces of polarized human primary PTEC under inflammatory diseased conditions. PTEC were cultured under normal and inflammatory conditions on Transwell inserts to enable separate collection and isolation of apical/basolateral sEV. Significantly increased numbers of apical and basolateral sEV were secreted under inflammatory conditions compared with equivalent normal conditions. Multi-omics analysis revealed distinct molecular profiles (lipids, microRNA, proteins) between inflammatory and normal conditions for both apical and basolateral sEV. Biological pathway analyses of significantly differentially expressed molecules associated apical inflammatory sEV with processes of cell survival and immunological disease, while basolateral inflammatory sEV were linked to pathways of immune cell trafficking and cell-to-cell signalling. In line with this mechanistic concept, functional assays demonstrated significantly increased production of chemokines (monocyte chemoattractant protein-1, interleukin-8) and immuno-regulatory cytokine interleukin-10 by peripheral blood mononuclear cells activated with basolateral sEV derived from inflammatory PTEC. We propose that the distinct molecular composition of sEV released from the apical versus basolateral membranes of human inflammatory PTEC may reflect specialized functional roles, with basolateral-derived sEV pivotal in modulating tubulointerstitial inflammatory responses observed in many immune-mediated kidney diseases. These findings provide a rationale to further evaluate these sEV-mediated inflammatory pathways as targets for biomarker and therapeutic development.

Marizomib suppresses triple-negative breast cancer via proteasome and oxidative phosphorylation inhibition.

Purpose: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required. Methods: We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo. Results: We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity. Conclusions: Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.

The α1-adrenoceptor inhibitor ρ-TIA facilitates net hunting in piscivorous Conus tulipa.

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

Transcriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, Conus imperialis.

Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis. Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.

Venomics Reveals Venom Complexity of the Piscivorous Cone Snail, Conus tulipa.

The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus, and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, µ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.

Headspace-free setup of in vitro bioassays for the evaluation of volatile disinfection by-products.

The conventional setup of in vitro bioassays in microplates does not prevent the loss of volatile compounds, which hampers the toxicological characterization of waterborne volatile disinfection by-products (DBPs). To minimize the loss of volatile test chemicals, we adapted four in vitro bioassays to a headspace-free setup using eight volatile organic compounds (four trihalomethanes, 1,1-dichloroethene, bromoethane, and two haloacetonitriles) that cover a wide range of air-water partition coefficients. The nominal effect concentrations of the test chemicals decreased by up to three orders of magnitude when the conventional setup was changed to a headspace-free setup for the bacterial cytotoxicity assay using bioluminescence inhibition of Vibrio fischeri. The increase of apparent sensitivity correlated significantly with the air-water partition coefficient. Purge and trap GC/MS analysis revealed a reduced loss of dosed volatile compounds in the headspace free setup (78-130% of nominal concentration) compared to a substantial loss in the conventional set up (2-13% of the nominal concentration). The experimental effect concentrations converged with the headspace-free setup to the effect concentrations predicted by a QSAR model, confirming the suitability of the headspace-free approach to minimize the loss of volatile test chemicals. The analogue headspace-free design of the bacterial bioassays for genotoxicity (umuC assay) and mutagenicity (Ames fluctuation assay) increased the number of compounds detected as genotoxic or mutagenic from one to four and zero to two, respectively. In a bioassay with a mammalian cell line applied for detecting the induction of the Nrf-2-mediated oxidative stress response (AREc32 assay), the headspace-free setup improved the apparent sensitivity by less than one order of magnitude, presumably due to the retaining effect of the serum components in the medium, which is also reflected in the reduced aqueous concentrations of compounds. This study highlights the importance of adapting bioanalytical test setups when volatile/semivolatile compounds are present in the sample to avoid the loss of chemicals and thus to avoid underestimating the toxicity of mixtures and complex environmental samples.

Most oxidative stress response in water samples comes from unknown chemicals: the need for effect-based water quality trigger values.

The induction of adaptive stress response pathways is an early and sensitive indicator of the presence of chemical and non-chemical stressors in cells. An important stress response is the Nrf-2 mediated oxidative stress response pathway where electrophilic chemicals or chemicals that cause the formation of reactive oxygen species initiate the production of antioxidants and metabolic detoxification enzymes. The AREc32 cell line is sensitive to chemicals inducing oxidative stress and has been previously applied for water quality monitoring of organic micropollutants and disinfection byproducts. Here we propose an algorithm for the derivation of effect-based water quality trigger values for this end point that is based on the combined effects of mixtures of regulated chemicals. Mixture experiments agreed with predictions by the mixture toxicity concept of concentration addition. The responses in the AREc32 and the concentrations of 269 individual chemicals were quantified in nine environmental samples, ranging from treated effluent, recycled water, stormwater to drinking water. The effects of the detected chemicals could explain less than 0.1% of the observed induction of the oxidative stress response in the sample, affirming the need to use effect-based trigger values that account for all chemicals present.

Water quality assessment using the AREc32 reporter gene assay indicative of the oxidative stress response pathway.

The reporter gene assay AREc32 is based on the induction of the Nrf2 mediated oxidative stress response pathway in the human breast cancer cell line MCF7, where eight copies of the antioxidant response element (ARE) are linked to a reporter gene encoding for luciferase. The Nrf2-ARE pathway is responsive to many chemicals that cause oxidative stress, among them a large number of pesticides and skin irritants. We adopted and validated the AREc32 bioassay for water quality testing. tert-Butylhydroquinone served as the positive control, phenol as the negative control and other reactive chemicals were assessed for their specificity. An environmentally relevant reference chemical, benzo(a)pyrene was the most potent inducer of all tested chemicals. The concentration causing an induction ratio (IR) of 1.5 (EC(IR1.5)) was chosen as the effect benchmark value. The assay was applied to 21 water samples ranging from sewage to drinking water, including secondary treatment and various tertiary treatment options (ozonation, biologically activated carbon filtration, membrane filtration, reverse osmosis, advanced oxidation, chlorination, chloramination). The samples were enriched by solid phase extraction. In most samples the oxidative stress response was far more sensitive than cytotoxicity. The primary and secondary treated effluent exceeded the effect threshold IR 1.5 at a relative enrichment factor (REF) of 1, i.e., the native samples were active. All tertiary treated samples were less potent and their EC(IR1.5) lay between REF 1 and 10. The Nrf2 pathway was induced at a REF of approximately 10 for surface waters and drinking water, and above this enrichment cytotoxicity took over in most samples and quenched the induction. The blank (ultrapure water run through the sample enrichment process) was cytotoxic at an REF of 100, which is the limit of concentrations range that can be evaluated. Treatment typically decreased both the cytotoxicity and oxidative stress response apart from drinking water treatment where chlorination caused an increase in oxidative stress response, presumably due to the formation of disinfection by-products. This study demonstrates the relevance and applicability of the oxidative stress response pathway for water quality monitoring.