Showing papers by "Steven P. Gygi published in 2020"

Dual Proteome-scale Networks Reveal Cell-specific Remodeling of the Human Interactome

Abstract: SUMMARY Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins – half the proteome – in 293T cells and includes 118,162 interactions among 14,586 proteins; the second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome at unprecedented scale, encoding protein function, localization, and complex membership. Their comparison validates thousands of interactions and reveals extensive customization of each network. While shared interactions reside in core complexes and involve essential proteins, cell-specific interactions bridge conserved complexes, likely ‘rewiring’ each cell’s interactome. Interactions are gained and lost in tandem among proteins of shared function as the proteome remodels to produce each cell’s phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.

TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples.

Abstract: Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs—all with essentially no missing values across the 16 samples and no loss in quantitative integrity. A set of isobaric labeling reagents called TMTpro enables deep quantitative comparisons of proteome measurements across 16 samples.

Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

Abstract: Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1–4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and β subunits that can no longer be phosphorylated by protein kinase A—an essential downstream mediator of β-adrenergic signalling—suggesting that non-channel factors are also required. Here we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or β2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels. An in vivo approach to identify proteins whose enrichment near cardiac CaV1.2 channels changes upon β-adrenergic stimulation finds the G protein Rad, which is phosphorylated by protein kinase A, thereby relieving channel inhibition by Rad and causing an increased Ca2+ current.

Full-featured, real-time database searching platform enables fast and accurate multiplexed quantitative proteomics

Abstract: Multiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. We built a new, real-time database search (RTS) platform, Orbiter, to combat the SPS-MS3 method's longer duty cycles. RTS with Orbiter eliminates SPS-MS3 scans if no peptide matches to a given spectrum. With Orbiter's online proteomic analytical pipeline, which includes RTS and false discovery rate analysis, it was possible to process a single spectrum database search in less than 10 ms. The result is a fast, functional means to identify peptide spectral matches using Comet, filter these matches, and more efficiently quantify proteins of interest. Importantly, the use of Comet for peptide spectral matching allowed for a fully featured search, including analysis of post-translational modifications, with well-known and extensively validated scoring. These data could then be used to trigger subsequent scans in an adaptive and flexible manner. In this work we tested the utility of this adaptive data acquisition platform to improve the efficiency and accuracy of multiplexed quantitative experiments. We found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency. Orbiter's RTS quantified more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 h for RTS, 36 h for SPS-MS3).

Selective Alanine Transporter Utilization Creates a Targetable Metabolic Niche in Pancreatic Cancer

Abstract: Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSC). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine cross-talk between PSCs and PDAC is orchestrated by the utilization of specific transporters. PSCs utilize SLC1A4 and other transporters to rapidly exchange and maintain environmental alanine concentrations. Moreover, PDAC cells upregulate SLC38A2 to supply their increased alanine demand. Cells lacking SLC38A2 fail to concentrate intracellular alanine and undergo a profound metabolic crisis resulting in markedly impaired tumor growth. Our results demonstrate that stromal-cancer metabolic niches can form through differential transporter expression, creating unique therapeutic opportunities to target metabolic demands of cancer. SIGNIFICANCE: This work identifies critical neutral amino acid transporters involved in channeling alanine between pancreatic stellate and PDAC cells. Targeting PDAC-specific alanine uptake results in a metabolic crisis impairing metabolism, proliferation, and tumor growth. PDAC cells specifically activate and require SLC38A2 to fuel their alanine demands that may be exploited therapeutically.This article is highlighted in the In This Issue feature, p. 890.

Defective NADPH production in mitochondrial disease complex I causes inflammation and cell death.

Abstract: Electron transport chain (ETC) defects occurring from mitochondrial disease mutations compromise ATP synthesis and render cells vulnerable to nutrient and oxidative stress conditions. This bioenergetic failure is thought to underlie pathologies associated with mitochondrial diseases. However, the precise metabolic processes resulting from a defective mitochondrial ETC that compromise cell viability under stress conditions are not entirely understood. We design a whole genome gain-of-function CRISPR activation screen using human mitochondrial disease complex I (CI) mutant cells to identify genes whose increased function rescue glucose restriction-induced cell death. The top hit of the screen is the cytosolic Malic Enzyme (ME1), that is sufficient to enable survival and proliferation of CI mutant cells under nutrient stress conditions. Unexpectedly, this metabolic rescue is independent of increased ATP synthesis through glycolysis or oxidative phosphorylation, but dependent on ME1-produced NADPH and glutathione (GSH). Survival upon nutrient stress or pentose phosphate pathway (PPP) inhibition depends on compensatory NADPH production through the mitochondrial one-carbon metabolism that is severely compromised in CI mutant cells. Importantly, this defective CI-dependent decrease in mitochondrial NADPH production pathway or genetic ablation of SHMT2 causes strong increases in inflammatory cytokine signatures associated with redox dependent induction of ASK1 and activation of stress kinases p38 and JNK. These studies find that a major defect of CI deficiencies is decreased mitochondrial one-carbon NADPH production that is associated with increased inflammation and cell death. Mitochondrial oxidative phosphorylation produces ATP and is an important source for cellular energy equivalents. Here the authors perform a cell-based screen to identify genes that alleviate perturbed mitochondrial complex I function and identify malic enzyme (ME-1), which promotes survival by production of NADPH and glutathione.

Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer's Disease Patients: A Pilot Study.

Abstract: Pathological hallmarks of Alzheimer's disease (AD) are deposits of amyloid beta (Aβ) and hyper-phosphorylated tau aggregates in brain plaques. Recent studies have highlighted the importance of Aβ and tau-containing extracellular vesicles (EVs) in AD. We therefore examined EVs separated from cerebrospinal fluid (CSF) of AD, mild cognitive impairment (MCI), and control (CTRL) patient samples to profile the protein composition of CSF EV. EV fractions were separated from AD (n = 13), MCI (n = 10), and CTRL (n = 10) CSF samples using MagCapture Exosome Isolation kit. The CSF-derived EV proteins were identified and quantified by label-free and tandem mass tag (TMT)-labeled mass spectrometry. Label-free proteomics analysis identified 2546 proteins that were significantly enriched for extracellular exosome ontology by Gene Ontology analysis. Canonical Pathway Analysis revealed glia-related signaling. Quantitative proteomics analysis, moreover, showed that EVs expressed 1284 unique proteins in AD, MCI and CTRL groups. Statistical analysis identified three proteins-HSPA1A, NPEPPS, and PTGFRN-involved in AD progression. In addition, the PTGFRN showed a moderate correlation with amyloid plaque (rho = 0.404, p = 0.027) and tangle scores (rho = 0.500, p = 0.005) in AD, MCI and CTRL. Based on the CSF EV proteomics, these data indicate that three proteins, HSPA1A, NPEPPS and PTGFRN, may be used to monitor the progression of MCI to AD.

Sample multiplexing for targeted pathway proteomics in aging mice

Abstract: Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted tandem mass tags-based sample multiplexing, hundreds of proteins can be quantified across 10 or more samples simultaneously. To facilitate these highly complex experiments, we introduce a strategy that provides complete control over targeted sample multiplexing experiments, termed Tomahto, and present its implementation on the Orbitrap Tribrid mass spectrometer platform. Importantly, this software monitors via the external desktop computer to the data stream and inserts optimized MS2 and MS3 scans in real time based on an application programming interface with the mass spectrometer. Hundreds of proteins of interest from diverse biological samples can be targeted and accurately quantified in a sensitive and high-throughput fashion. It achieves sensitivity comparable to, if not better than, deep fractionation and requires minimal total sample input (∼10 µg). As a proof-of-principle experiment, we selected four pathways important in metabolism- and inflammation-related processes (260 proteins/520 peptides) and measured their abundance across 90 samples (nine tissues from five old and five young mice) to explore effects of aging. Tissue-specific aging is presented here and we highlight the role of inflammation- and metabolism-related processes in white adipose tissue. We validated our approach through comparison with a global proteome survey across the tissues, work that we also provide as a general resource for the community.

High-density chemical cross-linking for modeling protein interactions.

Abstract: Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate. This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are self-consistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique cross-linked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.

Optimized Workflow for Multiplexed Phosphorylation Analysis of TMT-Labeled Peptides Using High-Field Asymmetric Waveform Ion Mobility Spectrometry.

Abstract: Phosphorylation is a post-translational modification with a vital role in cellular signaling. Isobaric labeling-based strategies, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the low stoichiometry of protein phosphorylation constrains the depth of phosphopeptide analysis by mass spectrometry. As such, robust and sensitive workflows are required. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT-labeled phosphopeptides. We determined that using FAIMS-MS3 with three compensation voltages (CV) in a single method (e.g., CV = -40/-60/-80 V) maximizes phosphopeptide coverage while minimizing inter-CV overlap. Furthermore, consecutive analyses using MSA-CID (multistage activation collision-induced dissociation) and HCD (higher-energy collisional dissociation) fragmentation at the MS2 stage increases the depth of phosphorylation analysis. The methodology and results outlined herein provide a template for tailoring optimized FAIMS-based methods.

O-GlcNAc regulates gene expression by controlling detained intron splicing.

Abstract: Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here, we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell's transcriptional regime.

Quantitative analysis of Y-Chromosome gene expression across 36 human tissues

Abstract: Little is known about how human Y-Chromosome gene expression directly contributes to differences between XX (female) and XY (male) individuals in nonreproductive tissues. Here, we analyzed quantitative profiles of Y-Chromosome gene expression across 36 human tissues from hundreds of individuals. Although it is often said that Y-Chromosome genes are lowly expressed outside the testis, we report many instances of elevated Y-Chromosome gene expression in a nonreproductive tissue. A notable example is EIF1AY, which encodes eukaryotic translation initiation factor 1A Y-linked, together with its X-linked homolog EIF1AX Evolutionary loss of a Y-linked microRNA target site enabled up-regulation of EIF1AY, but not of EIF1AX, in the heart. Consequently, this essential translation initiation factor is nearly twice as abundant in male as in female heart tissue at the protein level. Divergence between the X and Y Chromosomes in regulatory sequence can therefore lead to tissue-specific Y-Chromosome-driven sex biases in expression of critical, dosage-sensitive regulatory genes.

Mitochondrial fatty acid synthesis coordinates oxidative metabolism in mammalian mitochondria.

Abstract: Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.

Benchmarking the Orbitrap Tribrid Eclipse for Next Generation Multiplexed Proteomics

Abstract: The rise of sample multiplexing in quantitative proteomics for the dissection of complex phenotypic comparisons has been advanced by the development of ever more sensitive and robust instrumentation. Here, we evaluated the utility of the Orbitrap Eclipse Tribrid mass spectrometer (advanced quadrupole filter, optimized FTMS scan overhead) and new instrument control software features (Precursor Fit filtering, TurboTMT and Real-time Peptide Search filtering). Multidimensional comparisons of these novel features increased total peptide identifications by 20% for SPS-MS3 methods and 14% for HRMS2 methods. Importantly Real-time Peptide Search filtering enabled a ∼2× throughput improvement for quantification. Across the board, these sensitivity increases were attained without sacrificing quantitative accuracy. New hardware and software features enable more efficient characterization in pursuit of comparative whole proteome insights.

Co-option of Plasmodium falciparum PP1 for egress from host erythrocytes.

Abstract: Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells. Plasmodium protein phosphatase PP1 is essential for the asexual proliferation of malaria parasites. Here the authors show that PP1 regulates egress of parasites from host red blood cells, integrating parasite intrinsic pathways with environmental signals for release into the bloodstream.

Chromatin accessibility promotes hematopoietic and leukemia stem cell activity

Abstract: Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.

Facultative protein selenation regulates redox sensitivity, adipose tissue thermogenesis, and obesity

Abstract: Oxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenocysteines, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of proteogenic selenocysteines are limited. Here we developed a mass spectrometric method to interrogate incorporation of selenium into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium as selenocysteine or selenomethionine into proteins that lack canonical encoding for selenocysteine. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including as selenocysteine-replacing cysteine at position 253 in uncoupling protein 1 (UCP1). This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1, elevated energy expenditure through thermogenic adipose tissue, and protected against obesity. Together, these findings reveal the existence of facultative protein selenation, which correlates with impacts on thermogenic adipocyte function and presumably other biological processes as well.

Selection of Heating Temperatures Improves the Sensitivity of the Proteome Integral Solubility Alteration Assay

Abstract: The thermal shift assay is a robust method of discovering protein-ligand interactions by measuring the alterations in protein thermal stability under various conditions. Several thermal shift assays have been developed and their throughput has been advanced greatly by the rapid progress in tandem mass tag-based quantitative proteomics. A recent paper by Gaetani et al. ( J. Proteome Res. 2019, 18 (11), 4027-4037) introduced the proteome integral solubility alteration (PISA) assay, further increasing throughput and simplifying the data analysis. Both ΔSm (a proxy of the difference between areas under the melting curves) and fold changes (ratios between integral samples) are readouts of the PISA assay and positively related to ΔTm (shift in melting temperatures). Here, we show that the magnitudes of these readouts are inherently small in PISA assay, which is a challenge for quantitation. Both simulation and experimental results show that the selection of a subset of heating temperatures ameliorates the small difference problem and improves the sensitivity of the PISA assay.

O-GlcNAc regulates gene expression by controlling detained intron splicing

Abstract: Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell’s transcriptional regime.

A conserved RNA degradation complex required for spreading and epigenetic inheritance of heterochromatin

Abstract: Heterochromatic domains containing histone H3 lysine 9 methylation (H3K9me) can be epigenetically inherited independently of underlying DNA sequence. To gain insight into the mechanisms that mediate epigenetic inheritance, we used a Schizosaccharomyces pombe inducible heterochromatin formation system to perform a genetic screen for mutations that abolish heterochromatin inheritance without affecting its establishment. We identified mutations in several pathways, including the conserved and essential Rix1-associated complex (henceforth the rixosome), which contains RNA endonuclease and polynucleotide kinase activities with known roles in ribosomal RNA processing. We show that the rixosome is required for spreading and epigenetic inheritance of heterochromatin in fission yeast. Viable rixosome mutations that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.