Timothy J. Griffin

Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40

Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Here, we identify PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt–mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.

Complementary Profiling of Gene Expression at the Transcriptome and Proteome Levels in Saccharomyces cerevisiae

Using an integrated genomic and proteomic approach, we have investigated the effects of carbon source perturbation on steady-state gene expression in the yeast Saccharomyces cerevisiae growing on either galactose or ethanol. For many genes, significant differences between the abundance ratio of the messenger RNA transcript and the corresponding protein product were observed. Insights into the perturbative effects on genes involved in respiration, energy generation, and protein synthesis were obtained that would not have been apparent from measurements made at either the messenger RNA or protein level alone, illustrating the power of integrating different types of data obtained from the same sample for the comprehensive characterization of biological systems and processes.

Complementary Profiling of Gene Expression at the Transcriptome and Proteome Levels in S. cerevisiae

Using an integrated genomic and proteomic approach, we have investigated the effects of carbon source perturbation on steady-state gene expression in the yeast Saccharomyces cerevisiae growing on either galactose or ethanol. For many genes, significant differences between the abundance ratio of the messenger RNA transcript and the corresponding protein product were observed. Insights into the perturbative effects on genes involved in respiration, energy generation, and protein synthesis were obtained that would not have been apparent from measurements made at either the messenger RNA or protein level alone, illustrating the power of integrating different types of data obtained from the same sample for the comprehensive characterization of biological systems and processes.

Oxidative stress and covalent modification of protein with bioactive aldehydes.

The term “oxidative stress” links the production of reactive oxygen species to a variety of metabolic outcomes, including insulin resistance, immune dysfunction, and inflammation. Antioxidant defense systems down-regulated due to disease and/or aging result in oxidatively modified DNA, carbohydrates, proteins, and lipids. Increased production of hydroxyl radical leads to the formation of lipid hydroperoxides that produce a family of α,β-unsaturated aldehydes. Such reactive aldehydes are subject to Michael addition reactions with the side chains of lysine, histidine, and cysteine residues, referred to as “protein carbonylation.” Although not widely appreciated, reactive lipids can accumulate to high levels in cells, resulting in extensive protein modification leading to either loss or gain of function. The use of mass spectrometric methods to identify the site and extent of protein carbonylation on a proteome-wide scale has expanded our view of how oxidative stress can regulate cellular processes.

Single-nucleotide Polymorphism Analysis by MALDI-TOF Mass Spectrometry

Single-nucleotide polymorphisms (SNPs) have great potential for use in genetic-mapping studies, which locate and characterize genes that are important in human disease and biological function. For SNPs to realize their full potential in genetic analysis, thousands of different SNP loci must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs, with future prospects for use in genetic analysis.

Carbonylation of Adipose Proteins in Obesity and Insulin Resistance Identification of Adipocyte Fatty Acid-binding Protein as a Cellular Target of 4-Hydroxynonenal

Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a ∼2–3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased ∼3–4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1α-1 (eEF1α1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids ∼10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.

PRR5, a Novel Component of mTOR Complex 2, Regulates Platelet-derived Growth Factor Receptor β Expression and Signaling

The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2, relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5 (PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells, A., and Rustgi, A. K. (2005) Genomics 85, 338–351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates with reduction in the expression level of platelet-derived growth factor receptor β (PDGFRβ). PRR5 silencing impairs PDGF-stimulated phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRβ expression and signaling.

A Catalogue of Human Saliva Proteins Identified by Free Flow Electrophoresis-based Peptide Separation and Tandem Mass Spectrometry

Human saliva has great potential for clinical disease diagnostics. Constructing a comprehensive catalogue of saliva proteins using proteomic approaches is a necessary first step to identifying potential protein biomarkers of disease. However, because of the challenge presented in cataloguing saliva proteins with widely varying abundance, new proteomic approaches are needed. To this end, we used a newly developed approach coupling peptide separation using free flow electrophoresis with linear ion trap tandem mass spectrometry to identify proteins in whole human saliva. We identified 437 proteins with high confidence (false positive rate below 1%), producing the largest catalogue of proteins from a single saliva sample to date and providing new information on the composition and potential diagnostic utility of this fluid. The statistically validated, transparently presented, and annotated dataset provides a model for presenting large scale proteomic data of this type, which should facilitate better dissemination and easier comparisons of proteomic datasets from future studies in saliva.

A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva

Comprehensive identification of proteins in whole human saliva is critical for appreciating its full diagnostic potential. However, this is challenged by the large dynamic range of protein abundance within the fluid. To address this problem, we used an analysis platform that coupled hexapeptide libraries for dynamic range compression (DRC) with three-dimensional (3D) peptide fractionation. Our approach identified 2340 proteins in whole saliva and represents the largest saliva proteomic dataset generated using a single analysis platform. Three-dimensional peptide fractionation involving sequential steps of preparative isoelectric focusing (IEF), strong cation exchange, and capillary reversed-phase liquid chromatography was essential for maximizing gains from DRC. Compared to saliva not treated with hexapeptide libraries, DRC substantially increased identified proteins across physicochemical and functional categories. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential for whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples.

Advances in quantitative proteomics using stable isotope tags

A great deal of current biological and clinical research is directed at the interpretation of the information contained in the human genome sequence in terms of the structure, function and control of biological systems and processes. Proteomics, the systematic analysis of proteins, is becoming a critical component in this endeavor because proteomic measurements are carried out directly on proteins--the catalysts and effectors of essentially all biological functions. To detect changes in protein profiles that might provide important diagnostic or functional insights, proteomic analyses necessarily have to be quantitative. This article summarizes recent technological advances in quantitative proteomics.