Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation.

Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance.

Insulin resistance in muscle, adipocytes and liver is a gateway to a number of metabolic diseases. Here, we show a selective deficiency in mitochondrial coenzyme Q (CoQ) in insulin-resistant adipose and muscle tissue. This defect was observed in a range of in vitro insulin resistance models and adipose tissue from insulin-resistant humans and was concomitant with lower expression of mevalonate/CoQ biosynthesis pathway proteins in most models.

The transcriptional response to oxidative stress is part of, but not sufficient for, insulin resistance in adipocytes.

Insulin resistance is a major risk factor for metabolic diseases such as Type 2 diabetes. Although the underlying mechanisms of insulin resistance remain elusive, oxidative stress is a unifying driver by which numerous extrinsic signals and cellular stresses trigger insulin resistance. Consequently, we sought to understand the cellular response to oxidative stress and its role in insulin resistance.

Metabolomic analysis of insulin resistance across different mouse strains and diets.

Insulin resistance is a major risk factor for many diseases. However, its underlying mechanism remains unclear in part because it is triggered by a complex relationship between multiple factors, including genes and the environment. Here, we used metabolomics combined with computational methods to identify factors that classified insulin resistance across individual mice derived from three different mouse strains fed two different diets.

Correction: Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

[This corrects the article DOI: 10.1371/journal.pone.

Cross-Species PTM Mapping from Phosphoproteomic Data.

Protein post-translational modifications (PTMs) are crucial for signal transduction in cells. In order to understand key cell signaling events, identification of functionally important PTMs, which are more likely to be evolutionarily conserved, is necessary. In recent times, high-throughput mass spectrometry (MS) has made quantitative datasets in diverse species readily available, which has led to a growing need for tools to facilitate cross-species comparison of PTM data.

Skeletal muscle extracellular matrix remodeling after short-term overfeeding in healthy humans.

Background: Skeletal muscle extracellular matrix (ECM) remodeling has been proposed as a feature of the pathogenic milieu associated with obesity and metabolic dysfunction. The aim of the current study was to examine the timeline of this response and determine whether 3 and 28days of overfeeding alters markers of ECM turnover.

Methods: Forty healthy individuals were overfed by 1250kcal/day for 28days.

Hyperactivation of the Insulin Signaling Pathway Improves Intracellular Proteostasis by Coordinately Up-regulating the Proteostatic Machinery in Adipocytes.

Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy.

mTORC2 and AMPK differentially regulate muscle triglyceride content via Perilipin 3.

Objective: We have recently shown that acute inhibition of both mTOR complexes (mTORC1 and mTORC2) increases whole-body lipid utilization, while mTORC1 inhibition had no effect. Therefore, we tested the hypothesis that mTORC2 regulates lipid metabolism in skeletal muscle.

Methods: Body composition, substrate utilization and muscle lipid storage were measured in mice lacking mTORC2 activity in skeletal muscle (specific knockout of RICTOR (Ric mKO)).

Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured.

Integrated expression analysis of muscle hypertrophy identifies as a negative regulator of muscle mass.

The transforming growth factor-β (TGF-β) signaling network is a critical regulator of skeletal muscle mass and function and, thus, is an attractive therapeutic target for combating muscle disease, but the underlying mechanisms of action remain undetermined. We report that follistatin-based interventions (which modulate TGF-β network activity) can promote muscle hypertrophy that ameliorates aging-associated muscle wasting. However, the muscles of old sarcopenic mice demonstrate reduced response to follistatin compared with healthy young-adult musculature.

Highlights from the 11th ISCB Student Council Symposium 2015. Dublin, Ireland. 10 July 2015.

A1 Highlights from the eleventh ISCB Student Council Symposium 2015 Katie Wilkins, Mehedi Hassan, Margherita Francescatto, Jakob Jespersen, R. Gonzalo Parra, Bart Cuypers, Dan DeBlasio, Alexander Junge, Anupama Jigisha, Farzana Rahman O1 Prioritizing a drug’s targets using both gene expression and structural similarity Griet Laenen, Sander Willems, Lieven Thorrez, Yves Moreau O2 Organism specific protein-RNA recognition: A computational analysis of protein-RNA complex structures from different organisms Nagarajan Raju, Sonia Pankaj Chothani, C. Ramakrishnan, Masakazu Sekijima; M.

Skeletal muscle and plasma lipidomic signatures of insulin resistance and overweight/obesity in humans.

Objective: Alterations in lipids in muscle and plasma have been documented in insulin-resistant people with obesity. Whether these lipid alterations are a reflection of insulin resistance or obesity remains unclear.

Methods: Nondiabetic sedentary individuals not treated with lipid-lowering medications were studied (n = 51).

Cross-species gene expression analysis identifies a novel set of genes implicated in human insulin sensitivity.

Objective: Insulin resistance (IR) is one of the earliest predictors of type 2 diabetes. However, diagnosis of IR is limited. High fat fed mouse models provide key insights into IR.

Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates.

Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling.

Dataset from the global phosphoproteomic mapping of early mitotic exit in human cells.

The presence or absence of a phosphorylation on a substrate at any particular point in time is a functional readout of the balance in activity between the regulatory kinase and the counteracting phosphatase. Understanding how stable or short-lived a phosphorylation site is required for fully appreciating the biological consequences of the phosphorylation. Our current understanding of kinases and their substrates is well established; however, the role phosphatases play is less understood.

Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity.

PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

Background: Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data.

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins.

Targeted phosphoproteomics of insulin signaling using data-independent acquisition mass spectrometry.

A major goal in signaling biology is the establishment of high-throughput quantitative methods for measuring changes in protein phosphorylation of entire signal transduction pathways across many different samples comprising temporal or dose data or patient samples. Data-independent acquisition (DIA) mass spectrometry (MS) methods, which involve tandem MS scans that are collected independently of precursor ion information and then are followed by targeted searching for known peptides, may achieve this goal. We applied DIA-MS to systematically quantify phosphorylation of components in the insulin signaling network in response to insulin as well as in stimulated cells exposed to a panel of kinase inhibitors targeting key downstream effectors in the network.

Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs.

Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells.

Application of Drug-Perturbed Essential Dynamics/Molecular Dynamics (ED/MD) to Virtual Screening and Rational Drug Design.

We present here the first application of a new algorithm, essential dynamics/molecular dynamics (ED/MD), to the field of small molecule docking. The method uses a previously existing molecular dynamics (MD) ensemble of a protein or protein-drug complex to generate, with a very small computational cost, perturbed ensembles which represent ligand-induced binding site flexibility in a more accurate way than the original trajectory. The use of these perturbed ensembles in a standard docking program leads to superior performance than the same docking procedure using the crystal structure or ensembles obtained from conventional MD simulations as templates.

Identification of non-macrocyclic small molecule inhibitors against the NS3/4A serine protease of hepatitis C virus through in silico screening.

Drug discovery and design for inhibition of the Hepatitis C Virus (HCV) NS3/4A serine protease is a major challenge. The broad, shallow, and generally featureless nature of the active site makes it a difficult target for "hit" selection especially using standard docking programs. There are several macrocyclic NS3/4A protease inhibitors that have been approved or are in clinical trials to treat chronic HCV (alone or as combination therapy), but most of the current therapies for HCV infection have untoward side effects, indicating a continuing medical need for the discovery of novel therapeutics with improved efficacy.