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Type 2 diabetes is preceded by a defective insulin response, yet our knowledge of the precise mechanisms is incomplete. Here, we investigate how insulin resistance alters skeletal muscle signaling and how exercise partially counteracts this effect. We measured parallel phenotypes and phosphoproteomes of insulin-resistant (IR) and insulin-sensitive (IS) men as they responded to exercise and insulin (n = 19, 114 biopsies), quantifying over 12,000 phosphopeptides in each biopsy. Insulin resistance involves selective and time-dependent alterations to signaling, including reduced insulin-stimulated mTORC1 and non-canonical signaling responses. Prior exercise promotes insulin sensitivity even in IR individuals by "priming" a portion of insulin signaling prior to insulin infusion. This includes MINDY1 S441, which we show is an AKT substrate. We found that MINDY1 knockdown enhances insulin-stimulated glucose uptake in rat myotubes. This work delineates the signaling alterations in IR skeletal muscle and identifies MINDY1 as a regulator of insulin action., Competing Interests: Declaration of interests C.P. was an employee of Pfizer during the study, and the work was supported by a research grant from Pfizer, Inc. J.F.P.W. holds shares in Pfizer, Inc. and is currently a consultant at Pfizer Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the Greenlandic Inuit population, 4% are homozygous carriers of a genetic nonsense TBC1D4 p.Arg684Ter variant leading to loss of the muscle-specific isoform of TBC1D4 and an approximately tenfold increased risk of type 2 diabetes 1 . Here we show the metabolic consequences of this variant in four female and four male homozygous carriers and matched controls. An extended glucose tolerance test reveals prolonged hyperglycaemia followed by reactive hypoglycaemia in the carriers. Whole-body glucose disposal is impaired during euglycaemic-hyperinsulinaemic clamp conditions and associates with severe insulin resistance in skeletal muscle only. Notably, a marked reduction in muscle glucose transporter GLUT4 and associated proteins is observed. While metabolic regulation during exercise remains normal, the insulin-sensitizing effect of a single exercise bout is compromised. Thus, loss of the muscle-specific isoform of TBC1D4 causes severe skeletal muscle insulin resistance without baseline hyperinsulinaemia. However, physical activity can ameliorate this condition. These observations offer avenues for personalized interventions and targeted preventive strategies., (© 2024. The Author(s).)

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos ('microPhos'), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation., (© 2024. The Author(s).)

Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway's organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits., Competing Interests: Jv, SM, HC, AV, MP, JS, SM, MN, SH, DJ No competing interests declared, (© 2023, van Gerwen et al.)

A major challenge in mass spectrometry-based phosphoproteomics lies in identifying the substrates of kinases, as currently only a small fraction of substrates identified can be confidently linked with a known kinase. Machine learning techniques are promising approaches for leveraging large-scale phosphoproteomics data to computationally predict substrates of kinases. However, the small number of experimentally validated kinase substrates (true positive) and the high data noise in many phosphoproteomics datasets together limit their applicability and utility. Here, we aim to develop advanced kinase-substrate prediction methods to address these challenges. Using a collection of seven large phosphoproteomics datasets, and both traditional and deep learning models, we first demonstrate that a 'pseudo-positive' learning strategy for alleviating small sample size is effective at improving model predictive performance. We next show that a data resampling-based ensemble learning strategy is useful for improving model stability while further enhancing prediction. Lastly, we introduce an ensemble deep learning model ('SnapKin') by incorporating the above two learning strategies into a 'snapshot' ensemble learning algorithm. We propose SnapKin, an ensemble deep learning method, for predicting substrates of kinases from large-scale phosphoproteomics data. We demonstrate that SnapKin consistently outperforms existing methods in kinase-substrate prediction. SnapKin is freely available at https://github.com/PYangLab/SnapKin., (© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe coronavirus disease 2019 (COVID-19). To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR-Cas9-mediated knockout of ACE2, we demonstrated that angiotensin-converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but that further processing in lung cells required TMPRSS2, while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems., Competing Interests: Conflict of interests E.P. is a co-founder and scientific advisor and holds equity in, and N.C. has a paid position with, Dynomics, a biotechnology company focused on the development of heart failure therapeutics. J.M.P. is a co-founder and shareholder of Mogrify, Ltd., a cell therapy company., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

White adipose tissue is deposited mainly as subcutaneous adipose tissue (SAT), often associated with metabolic protection, and abdominal/visceral adipose tissue, which contributes to metabolic disease. To investigate the molecular underpinnings of these differences, we conducted comprehensive proteomics profiling of whole tissue and isolated adipocytes from these two depots across two diets from C57Bl/6J mice. The adipocyte proteomes from lean mice were highly conserved between depots, with the major depot-specific differences encoded by just 3% of the proteome. Adipocytes from SAT (SAdi) were enriched in pathways related to mitochondrial complex I and beiging, whereas visceral adipocytes (VAdi) were enriched in structural proteins and positive regulators of mTOR presumably to promote nutrient storage and cellular expansion. This indicates that SAdi are geared toward higher catabolic activity, while VAdi are more suited for lipid storage. By comparing adipocytes from mice fed chow or Western diet (WD), we define a core adaptive proteomics signature consisting of increased extracellular matrix proteins and decreased fatty acid metabolism and mitochondrial Coenzyme Q biosynthesis. Relative to SAdi, VAdi displayed greater changes with WD including a pronounced decrease in mitochondrial proteins concomitant with upregulation of apoptotic signaling and decreased mitophagy, indicating pervasive mitochondrial stress. Furthermore, WD caused a reduction in lipid handling and glucose uptake pathways particularly in VAdi, consistent with adipocyte de-differentiation. By overlaying the proteomics changes with diet in whole adipose tissue and isolated adipocytes, we uncovered concordance between adipocytes and tissue only in the visceral adipose tissue, indicating a unique tissue-specific adaptation to sustained WD in SAT. Finally, an in-depth comparison of isolated adipocytes and 3T3-L1 proteomes revealed a high degree of overlap, supporting the utility of the 3T3-L1 adipocyte model. These deep proteomes provide an invaluable resource highlighting differences between white adipose depots that may fine-tune their unique functions and adaptation to an obesogenic environment., Competing Interests: Conflict of interest The authors declare no competing interests, (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal-regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity., (© 2023. The Author(s).)

Spermatozoa acquire fertilization potential during passage through a highly specialized region of the extratesticular ductal system known as the epididymis. In the absence of de novo gene transcription or protein translation, this functional transformation is extrinsically driven via the exchange of varied macromolecular cargo between spermatozoa and the surrounding luminal plasma. Key among these changes is a substantive remodeling of the sperm proteomic architecture, the scale of which has yet to be fully resolved. Here, we have exploited quantitative mass spectrometry-based proteomics to define the extent of changes associated with the maturation of mouse spermatozoa; reporting the identity of >6,000 proteins, encompassing the selective loss and gain of several hundred proteins. Further, we demonstrate epididymal-driven activation of RHOA-mediated signaling pathways is an important component of sperm maturation. These data contribute molecular insights into the complexity of proteomic changes associated with epididymal sperm maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1 , Stx6 , Stx16 , Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types., (© 2022 Diaz-Vegas et al.)

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., (© 2022 The Author(s).)

Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce 'personalized phosphoproteomics', a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Cells respond to changing nutrient environments by adjusting the abundance of surface nutrient transporters and receptors. This can be achieved by modulating ubiquitin-dependent endocytosis, which in part is regulated by the NEDD4 family of E3 ligases. Here we report novel regulation of Pub1, a fission yeast Schizosaccharomyces pombe member of the NEDD4-family of E3 ligases. We show that nitrogen stress inhibits Pub1 function, thereby increasing the abundance of the amino acid transporter Aat1 at the plasma membrane and enhancing sensitivity to the toxic arginine analogue canavanine. We show that TOR complex 2 (TORC2) signalling negatively regulates Pub1, thus TORC2 mutants under nutrient stress have decreased Aat1 at the plasma membrane and are resistant to canavanine. Inhibition of TORC2 signalling increases Pub1 phosphorylation, and this is dependent on Gsk3 activity. Addition of the Tor inhibitor Torin1 increases phosphorylation of Pub1 at serine 199 (S199) by 2.5-fold, and Pub1 protein levels in S199A phospho-ablated mutants are reduced. S199 is conserved in NEDD4 and is located immediately upstream of a WW domain required for protein interaction. Together, we describe how the major TORC2 nutrient-sensing signalling network regulates environmental control of Pub1 to modulate the abundance of nutrient transporters., (© 2022 Wang et al.)

MaxDIA is a software platform for analyzing data-independent acquisition (DIA) proteomics data within the MaxQuant software environment. Using spectral libraries, MaxDIA achieves deep proteome coverage with substantially better coefficients of variation in protein quantification than other software. MaxDIA is equipped with accurate false discovery rate (FDR) estimates on both library-to-DIA match and protein levels, including when using whole-proteome predicted spectral libraries. This is the foundation of discovery DIA-hypothesis-free analysis of DIA samples without library and with reliable FDR control. MaxDIA performs three- or four-dimensional feature detection of fragment data, and scoring of matches is augmented by machine learning on the features of an identification. MaxDIA's bootstrap DIA workflow performs multiple rounds of matching with increasing quality of recalibration and stringency of matching to the library. Combining MaxDIA with two new technologies-BoxCar acquisition and trapped ion mobility spectrometry-both lead to deep and accurate proteome quantification., (© 2021. The Author(s).)

Pancreatic islets are essential for maintaining physiological blood glucose levels, and declining islet function is a hallmark of type 2 diabetes. We employ mass spectrometry-based proteomics to systematically analyze islets from 9 genetic or diet-induced mouse models representing a broad cross-section of metabolic health. Quantifying the islet proteome to a depth of >11,500 proteins, this study represents the most detailed analysis of mouse islet proteins to date. Our data highlight that the majority of islet proteins are expressed in all strains and diets, but more than half of the proteins vary in expression levels, principally due to genetics. Associating these varied protein expression levels on an individual animal basis with individual phenotypic measures reveals islet mitochondrial function as a major positive indicator of metabolic health regardless of strain. This compendium of strain-specific and dietary changes to mouse islet proteomes represents a comprehensive resource for basic and translational islet cell biology., Competing Interests: The authors declare no competing financial or non-financial interests., (© 2021 The Author(s).)

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., Competing Interests: AK, DN, MG, MK, SH, LC, GY, KC, PY, TG, SS, DF, LN, JB No competing interests declared, DJ Reviewing editor, eLife, (© 2021, Kearney et al.)

The selective and efficient capture of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis. Here we report a new switchable two-dimensional (2D) supramolecular polymer that serves as an ideal platform for the enrichment of phosphopeptides. A well-defined, positively charged metallacycle incorporated into the polymer endows the resultant polymer with a high affinity for phosphopeptides. Importantly, the stimuli-responsive nature of the polymer facilitates switchable binding affinity of phosphopeptides, thus resulting in an excellent performance in phosphopeptide enrichment and separation from model proteins. The polymer has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol), high enrichment recovery (88%), excellent specificity, and rapid enrichment and separation properties. Additionally, we have demonstrated the capture of phosphopeptides from the tryptic digest of real biosamples, thus illustrating the potential of this polymeric material in phosphoproteomic studies.

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.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1β, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage., Competing Interests: Declaration of interests R.J.M., J.E.H., G.A.Q.-R., D.M.T., and E.R.P. are co-inventors on patents relating to cardiac organoid maturation and cardiac therapeutics. J.E.H. is co-inventor on licensed patents for engineered heart muscle. R.J.M., E.R.P., D.M.T., B.G., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics. D.M.T. and B.G. are employees of Dynomics. C.H., D.G., L.F., J.J., M.S., N.C.W.W., and E.K. are employees of Resverlogix. S.J.N. received honoraria and research support from Resverlogix. QIMR Berghofer Medical Research Institute filed a patent on the use of BETi., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5' AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe . Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPK α ( ssp2::ura4 + ), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

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., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent advances in mass spectrometry (MS)-based quantitative proteomics now allow the identification and quantification of deep proteomes and post-translational modifications (PTMs) in relatively short times. Therefore, in the last few years, this technology has proven successful in the circadian field to characterize temporal oscillations of the proteome and more recently PTMs in cellular systems and in tissues. In this chapter, we describe a robust and simple protocol, based on the EasyPhos workflow, to enable preparation of large number of proteomes and phosphoproteomes from mouse tissues for MS-based quantitative analysis. We additionally discuss computational methods to analyze proteome and phosphoproteome time series to determine circadian oscillations.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Krycer et al.)

Sphingosine 1-phosphate (S1P) is an essential lipid metabolite that signals through a family of five G protein-coupled receptors, S1PR1-S1PR5, to regulate cell physiology. The multiple sclerosis drug Fingolimod (FTY720) is a potent S1P receptor agonist that causes peripheral lymphopenia. Recent research has demonstrated direct neuroprotective properties of FTY720 in several neurodegenerative paradigms; however, neuroprotective properties of the native ligand S1P have not been established. We aimed to establish the significance of neurotrophic factor up-regulation by S1P for neuroprotection, comparing S1P with FTY720. S1P induced brain-derived neurotrophic factor (BDNF), leukemia inhibitory factor (LIF), platelet-derived growth factor B (PDGFB), and heparin-binding EGF-like growth factor (HBEGF) gene expression in primary human and murine astrocytes, but not in neurons, and to a much greater extent than FTY720. Accordingly, S1P but not FTY720 protected cultured neurons against excitotoxic cell death in a primary murine neuron-glia coculture model, and a neutralizing antibody to LIF blocked this S1P-mediated neuroprotection. Antagonists of S1PR1 and S1PR2 both inhibited S1P-mediated neurotrophic gene induction in human astrocytes, indicating that simultaneous activation of both receptors is required. S1PR2 signaling was transduced through Gα 13 and the small GTPase Rho, and was necessary for the up-regulation and activation of the transcription factors FOS and JUN, which regulate LIF, BDNF, and HBEGF transcription. In summary, we show that S1P protects hippocampal neurons against excitotoxic cell death through up-regulation of neurotrophic gene expression, particularly LIF, in astrocytes. This up-regulation requires both S1PR1 and S1PR2 signaling. FTY720 does not activate S1PR2, explaining its relative inefficacy compared to S1P., (© 2019 International Society for Neurochemistry.)

The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved 'trans-omic' datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in the naive state, indicating that they are poised for expression prior to the initiation of pluripotency transition to the formative state. Finally, our reconstructed transcriptional networks pinpointed the precise timing from naive to formative pluripotency progression and enabled the spatiotemporal mapping of differentiating ESCs to their in vivo counterparts in developing embryos., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 PIP 3 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.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of β-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Limiting contamination of liquid chromatography/tandem mass spectrometry (LC-MS) systems and reducing the downtime associated with maintenance and cleaning is essential for productivity. We developed a simple device that creates a gas curtain barrier to prevent ions from entering the MS inlet. The gas can be quickly and easily applied when certain contaminant ions are known to elute. We show that the device can prevent the buildup of contaminants on the heated transfer capillary following >100 injections of a crude tissue lysate and improves peptide identifications. The device may provide a promising approach toward improving instrument robustness.

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/β-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 β-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 β-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., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The increasing role played by liquid chromatography-mass spectrometry (LC-MS)-based proteomics in biological discovery has led to a growing need for quality control (QC) on the LC-MS systems. While numerous quality control tools have been developed to track the performance of LC-MS systems based on a pre-defined set of performance factors (e.g., mass error, retention time), the precise influence and contribution of the performance factors and their generalization property to different biological samples are not as well characterized. Here, a web-based application (QCMAP) is developed for interactive diagnosis and prediction of the performance of LC-MS systems across different biological sample types. Leveraging on a standardized HeLa cell sample run as QC within a multi-user facility, predictive models are trained on a panel of commonly used performance factors to pinpoint the precise conditions to a (un)satisfactory performance in three LC-MS systems. It is demonstrated that the learned model can be applied to predict LC-MS system performance for brain samples generated from an independent study. By compiling these predictive models into our web-application, QCMAP allows users to benchmark the performance of their LC-MS systems using their own samples and identify key factors for instrument optimization. QCMAP is freely available from: http://shiny.maths.usyd.edu.au/QCMAP/., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Progressive decline of pancreatic beta cell function is central to the pathogenesis of type 2 diabetes. Protein phosphorylation regulates glucose-stimulated insulin secretion from beta cells, but how signaling networks are remodeled in diabetic islets in vivo remains unknown. Using high-sensitivity mass spectrometry-based proteomics, we quantified 6,500 proteins and 13,000 phosphopeptides in islets of obese diabetic mice and matched controls, revealing drastic remodeling of key kinase hubs and signaling pathways. Integration with a literature-derived signaling network implicated GSK3 kinase in the control of the beta cell-specific transcription factor PDX1. Deep phosphoproteomic analysis of human islets chronically treated with high glucose demonstrated a conserved glucotoxicity-dependent role of GSK3 kinase in regulating insulin secretion. Remarkably, the ability of beta cells to secrete insulin in response to glucose was rescued almost completely by pharmacological inhibition of GSK3. Thus, our resource enables investigation of mechanisms and drug targets in type 2 diabetes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem cell states. The two states that bookend the pluripotency continuum, naive and primed, are well characterized, but our understanding of the intermediate states and transitions between them remains incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre- to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naive to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of the kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell-state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlas.org)., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb and an amino acid-responsive pathway mediated via the Rag GTPases. Here, we identify and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. We find that RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation-induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, we identify mTORC1 as the upstream kinase of RagC on S21. Our data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity., (© 2018 The Authors.)

Protein phosphorylation is a major regulator of protein function and biological outcomes. This was first recognized through functional biochemical experiments, and in the past decade, major technological advances in mass spectrometry have enabled the study of protein phosphorylation on a global scale. This rapidly growing field of phosphoproteomics has revealed that more than 100,000 distinct phosphorylation events occur in human cells, which likely affect the function of every protein. Phosphoproteomics has improved the understanding of the function of even the most well-characterized protein kinases by revealing new downstream substrates and biology. However, current biochemical and bioinformatic approaches have only identified kinases for less than 5% of the phosphoproteome, and functional assignments of phosphosites are almost negligible. Notably, our understanding of the relationship between kinases and their substrates follows a power law distribution, with almost 90% of phosphorylation sites currently assigned to the top 20% of kinases. In addition, more than 150 kinases do not have a single known substrate. Despite a small group of kinases dominating biomedical research, the number of substrates assigned to a kinase does not correlate with disease relevance as determined by pathogenic human mutation prevalence and mouse model phenotypes. Improving our understanding of the substrates targeted by all kinases and functionally annotating the phosphoproteome will be broadly beneficial. Advances in phosphoproteomics technologies, combined with functional screening approaches, should make it feasible to illuminate the connectivity and functionality of the entire phosphoproteome, providing enormous opportunities for discovering new biology, therapeutic targets, and possibly diagnostics., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Insulin resistance is a pathophysiological state defined by impaired responses to insulin and is a risk factor for several metabolic diseases, most notably type 2 diabetes. Insulin resistance occurs in insulin target tissues including liver, adipose and skeletal muscle. Methods such as insulin tolerance tests and hyperinsulinaemic-euglycaemic clamps permit assessment of insulin responses in specific tissues and allow the study of the progression and causes of insulin resistance. Here we detail a protocol for assessing insulin action in adipose and muscle tissues in anesthetized mice administered with insulin intravenously., Competing Interests: Competing interestsThe authors have no competing interests to declare., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

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.

A systems view of G protein-coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)