Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (under chow and high fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

Human PIK3R1 mutations disrupt lymphocyte differentiation to cause activated PI3Kδ syndrome 2.

Heterozygous loss-of-function (LOF) mutations in PIK3R1 (encoding phosphatidylinositol 3-kinase [PI3K] regulatory subunits) cause activated PI3Kδ syndrome 2 (APDS2), which has a similar clinical profile to APDS1, caused by heterozygous gain-of-function (GOF) mutations in PIK3CD (encoding the PI3K p110δ catalytic subunit). While several studies have established how PIK3CD GOF leads to immune dysregulation, less is known about how PIK3R1 LOF mutations alter cellular function. By studying a novel CRISPR/Cas9 mouse model and patients' immune cells, we determined how PIK3R1 LOF alters cellular function. We observed some overlap in cellular defects in APDS1 and APDS2, including decreased intrinsic B cell class switching and defective Tfh cell function. However, we also identified unique APDS2 phenotypes including defective expansion and affinity maturation of Pik3r1 LOF B cells following immunization, and decreased survival of Pik3r1 LOF pups. Further, we observed clear differences in the way Pik3r1 LOF and Pik3cd GOF altered signaling. Together these results demonstrate crucial differences between these two genetic etiologies.

SILAC kinase screen identifies potential MASTL substrates.

Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.

Structural insights into Ras regulation by SIN1.

Over the years it has been established that SIN1, a key component of mTORC2, could interact with Ras family small GTPases through its Ras-binding domain (RBD). The physical association of Ras and SIN1/mTORC2 could potentially affect both mTORC2 and Ras-ERK pathways. To decipher the precise molecular mechanism of this interaction, we determined the high-resolution structures of HRas/KRas-SIN1 RBD complexes, showing the detailed interaction interface. Mutation of critical interface residues abolished Ras-SIN1 interaction and in SIN1 knockout cells we demonstrated that Ras-SIN1 association promotes SGK1 activity but inhibits insulin-induced ERK activation. With structural comparison and competition fluorescence resonance energy transfer (FRET) assays we showed that HRas-SIN1 RBD association is much weaker than HRas-Raf1 RBD but is slightly stronger than HRas-PI3K RBD interaction, providing a possible explanation for the different outcome of insulin or EGF stimulation. We also found that SIN1 isoform lacking the PH domain binds stronger to Ras than other longer isoforms and the PH domain appears to have an inhibitory effect on Ras-SIN1 binding. In addition, we uncovered a Ras dimerization interface that could be critical for Ras oligomerization. Our results advance our understanding of Ras-SIN1 association and crosstalk between growth factor-stimulated pathways.

Dynamic modelling of the PI3K/MTOR signalling network uncovers biphasic dependence of mTORC1 activity on the mTORC2 subunit SIN1.

The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GßL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2's activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis.

The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold.

For the body to work properly, cells must constantly 'talk' to each other using signalling molecules. Receiving a chemical signal triggers a series of molecular events in a cell, a so-called 'signal transduction pathway' that connects a signal with a precise outcome. Disturbing cell signalling can trigger disease, and strict control mechanisms are therefore in place to ensure that communication does not break down or become erratic. For instance, just as a thermostat turns off the heater once the right temperature is reached, negative feedback mechanisms in cells switch off signal transduction pathways when the desired outcome has been achieved. The hormone insulin is a signal for growth that increases in the body following a meal to promote the storage of excess blood glucose (sugar) in muscle and fat cells. The hormone binds to insulin receptors at the cell surface and switches on a signal transduction pathway that makes the cell take up glucose from the bloodstream. If the signal is not engaged diseases such as diabetes develop. Conversely, if the signal cannot be adequately switched of cancer can develop. Determining exactly how insulin works would help to understand these diseases better and to develop new treatments. Kearney et al. therefore set out to examine the biochemical 'fail-safes' that control insulin signalling. Experiments using computer simulations of the insulin signalling pathway revealed a potential new mechanism for negative feedback, which centred on a molecule known as Akt. The models predicted that if the negative feedback were removed, then Akt would become hyperactive and accumulate at the cell's surface after stimulation with insulin. Further manipulation of the 'virtual' insulin signalling pathway and studies of live cells in culture confirmed that this was indeed the case. The cell biology experiments also showed how Akt, once at the cell surface, was able to engage the negative feedback and shut down further insulin signalling. Akt did this by inactivating a protein required to pass the signal from the insulin receptor to the rest of the cell. Overall, this work helps to understand cell communication by revealing a previously unknown, and critical component of the insulin signalling pathway.

The aetiology and molecular landscape of insulin resistance.

Insulin resistance, defined as a defect in insulin-mediated control of glucose metabolism in tissues - prominently in muscle, fat and liver - is one of the earliest manifestations of a constellation of human diseases that includes type 2 diabetes and cardiovascular disease. These diseases are typically associated with intertwined metabolic abnormalities, including obesity, hyperinsulinaemia, hyperglycaemia and hyperlipidaemia. Insulin resistance is caused by a combination of genetic and environmental factors. Recent genetic and biochemical studies suggest a key role for adipose tissue in the development of insulin resistance, potentially by releasing lipids and other circulating factors that promote insulin resistance in other organs. These extracellular factors perturb the intracellular concentration of a range of intermediates, including ceramide and other lipids, leading to defects in responsiveness of cells to insulin. Such intermediates may cause insulin resistance by inhibiting one or more of the proximal components in the signalling cascade downstream of insulin (insulin receptor, insulin receptor substrate (IRS) proteins or AKT). However, there is now evidence to support the view that insulin resistance is a heterogeneous disorder that may variably arise in a range of metabolic tissues and that the mechanism for this effect likely involves a unified insulin resistance pathway that affects a distal step in the insulin action pathway that is more closely linked to the terminal biological response. Identifying these targets is of major importance, as it will reveal potential new targets for treatments of diseases associated with insulin resistance.

Global phosphoproteomics reveals DYRK1A regulates CDK1 activity in glioblastoma cells.

Both tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.

PhosR enables processing and functional analysis of phosphoproteomic data.

Mass spectrometry (MS)-based phosphoproteomics has revolutionized our ability to profile phosphorylation-based signaling in cells and tissues on a global scale. To infer the action of kinases and signaling pathways in phosphoproteomic experiments, we present PhosR, a set of tools and methodologies implemented in a suite of R packages facilitating comprehensive analysis of phosphoproteomic data. By applying PhosR to both published and new phosphoproteomic datasets, we demonstrate capabilities in data imputation and normalization by using a set of "stably phosphorylated sites" and in functional analysis for inferring active kinases and signaling pathways. In particular, we introduce a "signalome" construction method for identifying a collection of signaling modules to summarize and visualize the interaction of kinases and their collective actions on signal transduction. Together, our data and findings demonstrate the utility of PhosR in processing and generating biological knowledge from MS-based phosphoproteomic data.

Global redox proteome and phosphoproteome analysis reveals redox switch in Akt.

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.

Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser474; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model where Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.

A modified gas-trapping method for high-throughput metabolic experiments in Drosophila melanogaster.

Metabolism is often studied in animal models, with the Drosophila melanogaster fruit fly model offering ease of genetic manipulation and high-throughput studies. Fly metabolism is typically studied using end-point assays that are simple but destructive, and do not provide information on the utilization of specific nutrients. To address these limitations, we adapted existing gas-trapping protocols to measure the oxidation of radiolabeled substrates (such as glucose) in multi-well plates. This protocol is cost effective, simple, and offers precise control over experimental diet and measurement time, thus being amenable to high-throughput studies. Furthermore, it is nondestructive, enabling time-course experiments and multiplexing with other parameters. Overall, this protocol is useful for merging fly genetics with metabolic studies to understand whole organism responses to different macronutrients.

ABHD15 regulates adipose tissue lipolysis and hepatic lipid accumulation.

OBJECTIVE: Insulin suppresses adipose tissue lipolysis after a meal, playing a key role in metabolic homeostasis. This is mediated via the kinase Akt and its substrate phosphodiesterase 3B (PDE3B). Once phosphorylated and activated, PDE3B hydrolyses cAMP leading to the inactivation of cAMP-dependent protein kinase (PKA) and suppression of lipolysis. However, several gaps have emerged in this model. Here we investigated the role of the PDE3B-interacting protein, α/ß-hydrolase ABHD15 in this process. METHODS: Lipolysis, glucose uptake, and signaling were assessed in ABHD15 knock down and knock out adipocytes and fat explants in response to insulin and/or ß-adrenergic receptor agonist. Glucose and fatty acid metabolism were determined in wild type and ABHD15-/- littermate mice. RESULTS: Deletion of ABHD15 in adipocytes resulted in a significant defect in insulin-mediated suppression of lipolysis with no effect on insulin-mediated glucose uptake. ABHD15 played a role in suppressing PKA signaling as phosphorylation of the PKA substrate Perilipin-1 remained elevated in response to insulin upon ABHD15 deletion. ABHD15-/- mice had normal glucose metabolism but defective fatty acid metabolism: plasma fatty acids were elevated upon fasting and in response to insulin, and this was accompanied by elevated liver triglycerides upon ß-adrenergic receptor activation. This is likely due to hyperactive lipolysis as evident by the larger triglyceride depletion in brown adipose tissue in these mice. Finally, ABHD15 protein levels were reduced in adipocytes from mice fed a Western diet, further implicating this protein in metabolic homeostasis. CONCLUSIONS: Collectively, ABHD15 regulates adipocyte lipolysis and liver lipid accumulation, providing novel therapeutic opportunities for modulating lipid homeostasis in disease.

Reduced insulin action in muscle of high fat diet rats over the diurnal cycle is not associated with defective insulin signaling.

OBJECTIVE: Energy metabolism and insulin action follow a diurnal rhythm. It is therefore important that investigations into dysregulation of these pathways are relevant to the physiology of this diurnal rhythm. METHODS: We examined glucose uptake, markers of insulin action, and the phosphorylation of insulin signaling intermediates in muscle of chow and high fat, high sucrose (HFHS) diet-fed rats over the normal diurnal cycle. RESULTS: HFHS animals displayed hyperinsulinemia but had reduced systemic glucose disposal and lower muscle glucose uptake during the feeding period. Analysis of gene expression, enzyme activity, protein abundance and phosphorylation revealed a clear diurnal regulation of substrate oxidation pathways with no difference in Akt signaling in muscle. Transfection of a constitutively active Akt2 into the muscle of HFHS rats did not rescue diet-induced reductions in insulin-stimulated glucose uptake. CONCLUSIONS: These studies suggest that reduced glucose uptake in muscle during the diurnal cycle induced by short-term HFHS-feeding is not the result of reduced insulin signaling.

Proteomic Analysis of Human Plasma during Intermittent Fasting.

Intermittent fasting (IF) increases lifespan and decreases metabolic disease phenotypes and cancer risk in model organisms, but the health benefits of IF in humans are less clear. Human plasma derived from clinical trials is one of the most difficult sample sets to analyze using mass spectrometry-based proteomics due to the extensive sample preparation required and the need to process many samples to achieve statistical significance. Here, we describe an optimized and accessible device (Spin96) to accommodate up to 96 StageTips, a widely used sample preparation medium enabling efficient and consistent processing of samples prior to LC-MS/MS. We have applied this device to the analysis of human plasma from a clinical trial of IF. In this longitudinal study employing 8-weeks IF, we identified significant abundance differences induced by the IF intervention, including increased apolipoprotein A4 (APOA4) and decreased apolipoprotein C2 (APOC2) and C3 (APOC3). These changes correlated with a significant decrease in plasma triglycerides after the IF intervention. Given that these proteins have a role in regulating apolipoprotein particle metabolism, we propose that IF had a positive effect on lipid metabolism through modulation of HDL particle size and function. In addition, we applied a novel human protein variant database to detect common protein variants across the participants. We show that consistent detection of clinically relevant peptides derived from both alleles of many proteins is possible, including some that are associated with human metabolic phenotypes. Together, these findings illustrate the power of accessible workflows for proteomics analysis of clinical samples to yield significant biological insight.

High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform.

Mass spectrometry has transformed the field of cell signaling by enabling global studies of dynamic protein phosphorylation ('phosphoproteomics'). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these and is sufficiently streamlined to enable the analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated workflow that further ensures high performance in sample-limited conditions while also reducing sample preparation time. By eliminating protein precipitation steps and performing the entire protocol, including digestion, in a single 96-well plate, we now greatly minimize opportunities for sample loss and variability. This results in very high reproducibility and a small sample size requirement (≤200 µg of protein starting material). After cell culture or tissue collection, the protocol takes 1 d, whereas mass spectrometry measurements require ~1 h per sample. Applied to glioblastoma cells acutely treated with epidermal growth factor (EGF), EasyPhos quantified 20,132 distinct phosphopeptides from 200 µg of protein in less than 1 d of measurement time, revealing thousands of EGF-regulated phosphorylation events.

Membrane Topology of Trafficking Regulator of GLUT4 1 (TRARG1).

Trafficking regulator of GLUT4 1 (TRARG1) was recently identified to localize to glucose transporter type 4 (GLUT4) storage vesicles (GSVs) and to positively regulate GLUT4 trafficking. Our knowledge of TRARG1 structure and membrane topology is limited to predictive models, hampering efforts to further our mechanistic understanding of how it carries out its functions. Here, we use a combination of bioinformatics prediction tools and biochemical assays to define the membrane topology of the 173-amino acid mouse TRARG1. These analyses revealed that, contrary to the consensus prediction, the N-terminus is cytosolic and that a short segment at the C-terminus resides in the luminal/extracellular space. Based on our biochemical analyses including membrane association and antibody accessibility assays, we conclude that TRARG1 has one transmembrane domain (TMD) (145-172) and a re-entrant loop between residues 101 and 127.