714 research outputs found
Combining amine metabolomics and quantitative proteomics of cancer cells using derivatization with isobaric tags
No full textQuantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons
Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR
No full textLigand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases
Comprehensive temporal protein dynamics during the diauxic shift in saccharomyces cerevisiae
No full textYeast (Saccharomyces cerevisiae) has served as a key model system in biology and as a benchmark for “omics” technology. Although near-complete proteomes of log phase yeast have been measured, protein abundance in yeast is dynamic, particularly during the transition from log to stationary phase. Defining the dynamics of proteomic changes during this transition, termed the diauxic shift, is important to understand the basic biology of proliferative versus quiescent cells. Here, we perform temporal quantitative proteomics to fully capture protein induction and repression during the diauxic shift. Accurate and sensitive quantitation at a high temporal resolution and depth of proteome coverage was achieved using TMT10 reagents and LC-MS3 analysis on an Orbitrap Fusion tribrid mass spectrometer deploying synchronous precursor selection. Triplicate experiments were analyzed using the time-course R package and a simple template matching strategy was used to reveal groups of proteins with similar temporal patterns of protein induction and repression. Within these groups are functionally distinct types of proteins such as those of glyoxylate metabolism and many proteins of unknown function not previously associated with the diauxic shift (e.g. YNR034W-A and FMP16). We also perform a dual time-course experiment to determine Hap2-dependent proteins during the diauxic shift. These data serve as an important basic model for fermentative versus respiratory growth of yeast and other eukaryotes and are a benchmark for temporal quantitative proteomics.
The yeast proteome serves as a valuable model in systems biology (1) and as such has been used to gauge technological milestones in proteomics. In recent years, the depth of protein identification in logarithmically-growing yeast has expanded to near-comprehensiveness (>4000 identified proteins) (2–3). However, the yeast proteome is dynamic, and understanding regulatory networks requires a comprehensive grasp of the timing of induction or repression of specific sets of proteins. Proteome dynamics in yeast depend largely on substrate availability, the major of which is glucose. The diauxic shift, the transition from log phase growth on glucose to stationary phase upon glucose exhaustion, involves temporal coordination of protein regulation that is still not completely understood. Because log phase yeast resemble fermentative cancer cells, the diauxic shift is also considered important basic model for understanding the requirements of proliferative cell growth. As such, a landmark gene expression study of the diauxic shift at the transcript level (4) has served as an important resource in biology (5). However, despite recent progress, the dynamic nature of the diauxic shift at the proteome level has not been adequately explored. Doing so requires the ability to perform comprehensive quantitative proteomic analysis with a sufficient number of time-points to resolve the timing of protein induction or repression. Although temporal proteomic data changes have recently been reported within both log and stationary phase (6) using six-plex quantitative proteomics with TMT, and during the transition between log and stationary phase using a four-plex Neucode strategy, highly resolved proteomic data able to fully capture protein dynamics between the two states are lacking.
Several recent advances in mass spectrometry and multiplexed quantitative proteomics potentiate accurate measurements of highly resolved time courses at a comprehensive depth of proteome coverage. First, tandem mass tag technology (TMT)1 has been expanded to compare up to 10 samples simultaneously (TMT10) using isotopologue-containing reporter ions distinguishable by high-resolution mass spectrometry (7). TMT reagents work on the principle of isobaric tagging whereby their addition to a peptide maintains a nominal mass, but cleavable reporter ions are used to compare peptide abundance between samples (8). Second, although isobaric reporter ion tagging strategies have been plagued by interference from co-isolated peptides, using MS3 scans for reporter ion quantitation has been shown to ensure quantitative accuracy (9). Third, the MS3 approach has recently been enhanced by techniques to isolate multiple MS2 precursors (SPS) for greater MS3 scan sensitivity (10). These advancements, deployed on the Orbitrap Fusion tribrid mass spectrometer allow high temporal resolution (10 time points), proteomic depth (>4500 proteins), and quantitative accuracy (11). We here aimed to fully capture the intricate temporal proteome dynamics over the full diauxic shift at a comprehensive level using 10-plex TMT and LC-MS3.
The data we present here extend current understanding of protein dynamics during diauxic shift in yeast by virtue of the high temporal resolution and comprehensive depth achieved. Data were collected in triplicate allowing us to assess temporal significance, following which we applied a simple template-profile matching strategy to map the timing of induction or repression of proteins of specific functions in relation to glucose exhaustion. The data set also resolves the timing of the induction of many proteins of unknown function (e.g. FMP16, YNR034W-A, and TMA17) as well those not previously associated with the transition to stationary phase (e.g. YKL065W-A and PAI3). In an additional, dual time-course experiment, we show both high reproducibility between analyses of different strains and provide a putative list of proteins dependent on the respiratory growth-related transcription factor, Hap2. These include many known Hap2-dependent proteins such as those of glyoxylate metabolism and oxidative phosphorylation as well as several novel Hap2-dependent proteins supporting carnitine metabolism (e.g. AGP2). These data serve as a benchmark in temporal quantitative proteomics and are a valuable resource for the biological community in understanding fermentative versus respiratory metabolism in eukaryotic cells
Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome
No full textCentrosomes are conserved organelles that are essential for accurate cell division and cilium formation. A centrosome consists of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM) that is essential for the centrosome's function. In this study, we show that Sas-4 provides a scaffold for cytoplasmic complexes (named S-CAP), which include CNN, Asl and D-PLP, proteins that are all found in the centrosomes at the vicinity of the centriole. When Sas-4 is absent, nascent procentrioles are unstable and lack PCM, and functional centrosomes are not generated. When Sas-4 is mutated, so that it cannot form S-CAP complexes, centrosomes are present but with dramatically reduced levels of PCM. Finally, purified S-CAP complexes or recombinant Sas-4 can bind centrosomes stripped of PCM, whereas recombinant CNN or Asl cannot. In summary, PCM assembly begins in the cytosol where Sas-4 provides a scaffold for pre-assembled cytoplasmic complexes before tethering of the complexes in a centrosome.</p
Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase
No full textThe hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48
High-density chemical cross-linking for modeling protein interactions
No full text<p>XL search engine code</p>
<p>IMP code for de novo modeling of yeast proteasome regulatory particle from cross-linking data</p>
PHD3 loss in cancer enables metabolic reliance on fatty acid oxidation via deactivation of ACC2
No full textWhile much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition
An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis
No full textMycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens
Therapy-induced MHC I ligands shape neo-antitumor CD8 T cell responses during oncolytic virus-based cancer immunotherapy
No full textOncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies
Differential glutamate metabolism in proliferating and quiescent mammary epithelial cells
No full textMammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation
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