Showing papers by "Steven P. Gygi published in 2019"
TL;DR: It is demonstrated that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs.
157 citations
TL;DR: High-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications.
Abstract: Multiplexed, isobaric tagging methods are powerful techniques to increase throughput, precision, and accuracy in quantitative proteomics. The dynamic range and accuracy of quantitation, however, can be limited by coisolation of tag-containing peptides that release reporter ions and conflate quantitative measurements across precursors. Methods to alleviate these effects often lead to the loss of protein and peptide identifications through online or offline filtering of interference containing spectra. To alleviate this effect, high-Field Asymmetric-waveform Ion Mobility Spectroscopy (FAIMS) has been proposed as a method to reduce precursor coisolation and improve the accuracy and dynamic range of multiplex quantitation. Here we tested the use of FAIMS to improve quantitative accuracy using previously established TMT-based interference standards (triple-knockout [TKO] and Human-Yeast Proteomics Resource [HYPER]). We observed that FAIMS robustly improved the quantitative accuracy of both high-resolution MS2 (HRMS2) and synchronous precursor selection MS3 (SPS-MS3)-based methods without sacrificing protein identifications. We further optimized and characterized the main factors that enable robust use of FAIMS for multiplexed quantitation. We highlight these factors and provide method recommendations to take advantage of FAIMS technology to improve isobaric-tag-quantification moving forward.
125 citations
TL;DR: It is demonstrated that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties, demonstrating that context-dependent variations in signaling downstream of differentKRAS mutants drive the KRAS mutational pattern seen in cancer.
Abstract: KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor-induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties.See related commentary by Hobbs and Der, p. 696.This article is highlighted in the In This Issue feature, p. 681.
120 citations
TL;DR: This work highlights EBV-induced 1C as a potential therapeutic target and provides a new paradigm for viral onco-metabolism, which is highlighted in previous work on Burkitt, Hodgkin, and post-transplant B cell lymphomas.
96 citations
TL;DR: EF-hand calcium-binding domain-containing protein 9 (EFCAB9) is identified as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper.
96 citations
TL;DR: Assessment of the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows indicates that the RTS-MS3 approach provides dramatic performance improvements for quantitativemultiplexed experiments.
Abstract: Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS3 approach provides dramatic performance improvements for quantitative multiplexed experiments.
92 citations
TL;DR: This work identifies numerous stereoselective protein–fragment interactions in cells and shows that these interactions occur at functional sites on proteins from diverse classes, indicating that incorporating chirality into fully functionalized fragment libraries provides a robust and streamlined method to discover ligandable proteins in cells.
Abstract: A fundamental challenge in chemical biology and medicine is to understand and expand the fraction of the human proteome that can be targeted by small molecules. We recently described a strategy that integrates fragment-based ligand discovery with chemical proteomics to furnish global portraits of reversible small-molecule/protein interactions in human cells. Excavating clear structure-activity relationships from these 'ligandability' maps, however, was confounded by the distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments. Here, we describe a compelling solution to this problem by introducing a next-generation set of fully functionalized fragments differing only in absolute stereochemistry. Using these enantiomeric probe pairs, or 'enantioprobes', we identify numerous stereoselective protein-fragment interactions in cells and show that these interactions occur at functional sites on proteins from diverse classes. Our findings thus indicate that incorporating chirality into fully functionalized fragment libraries provides a robust and streamlined method to discover ligandable proteins in cells.
86 citations
TL;DR: In this paper, the RNA helicase DDX6 was found to influence the balance between self-renewal and differentiation in a context-dependent manner in adult stem cells.
70 citations
TL;DR: The microbiota can shape the post-translational landscape of the host proteome to regulate microRNA activity, gene expression, and host development, and suggest a general mechanism by which the microbiota may control host cellular functions, as well as a new role for gasotransmitters.
57 citations
TL;DR: It is concluded that false transfers by MBR are plentiful, but few are retained in the final dataset after applying the MaxQuant LFQ algorithm.
Abstract: Stochasticity between independent LC-MS/MS runs is a challenging problem in the field of proteomics, resulting in significant missing values (i.e., abundance measurements) among observed peptides. To address this issue, several approaches have been developed including computational methods such as MaxQuant's match-between-runs (MBR) algorithm. Often dozens of runs are all considered at once by MBR, transferring identifications from any one run to any of the others. To evaluate the error associated with these transfer events, we created a two-sample/two-proteome approach. In this way, samples containing no yeast lysate (n = 20) were assessed for false identification transfers from samples containing yeast (n = 20). While MBR increased the total number of spectral identifications by ∼40%, we also found that 44% of all identified yeast proteins had identifications transferred to at least one sample without yeast. However, of these only 2.7% remained in the final data set after applying the MaxQuant LFQ algorithm. We conclude that false transfers by MBR are plentiful, but few are retained in the final data set.
44 citations
TL;DR: Examination of the protein composition of EVs isolated from the cerebrospinal fluid of former National Football League players with cognitive and neuropsychiatric dysfunction revealed enrichment of canonical liver/retinoid X receptor activation pathway and upstream effect analysis predicted MAPT (tau) as an upstream regulator in former NFL players.
Abstract: Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of repetitive mild traumatic brain injury, such as American football players. Initial neuropathologic changes in CTE include perivascular deposition of phosphorylated microtubule-associated protein tau (p-tau) neurofibrillary tangles and other aggregates in neurons, astrocytes and cell processes in an irregular pattern often at the depths of the cortical sulci. In later stages, the p-tau depositions become widespread and is associated with neurodegeneration. Extracellular vesicles (EVs) are known to carry neuropathogenic molecules, most notably p-tau. We therefore examined the protein composition of EVs isolated from the cerebrospinal fluid (CSF) of former National Football League (NFL) players with cognitive and neuropsychiatric dysfunction, and an age-matched control group (CTRL) with no history of contact sports or traumatic brain injury. EVs were isolated from the CSF samples using an affinity purification kit. Total tau (t-tau) and tau phosphorylated on threonine181 (p-tau181) in CSF-derived EVs from former NFL players and CTRL participants were measured by ultrasensitive immunoassay. The t-tau and p-tau181 levels of CSF-derived EV were positively correlated with the t-tau and p-tau181 levels of total CSF in former NFL players, respectively, but not in the CTRL group. 429 unique proteins were identified from CSF-derived EVs and quantified by TMT-10 plex method. The identified protein molecules were significantly enriched for the extracellular exosome molecules, Alzheimer's disease pathway and Age/Telomere Length ontology as determined by DAVID Gene Ontology analysis. Ingenuity pathway analysis of the differentially expressed EV proteins revealed enrichment of canonical liver/retinoid X receptor activation pathway. Upstream effect analysis predicted MAPT (tau) as an upstream regulator in former NFL players. These data will be useful for understanding the EV-mediated disease spread and development of novel EV biomarkers for CTE and related disorders.
TL;DR: The TPX2/Aurora A heterodimer, nominally considered a mitotic kinase complex, as a novel binding partner of 53BP1 is identified and the presence of a feedback mechanism by which 53 BP1 is regulated by a novelbinding partner is suggested and a unique role for 53BP 1 in replication fork stability is uncovered.
Abstract: 53BP1 is a chromatin-associated protein that regulates the DNA damage response. In this study, we identify the TPX2/Aurora A heterodimer, nominally considered a mitotic kinase complex, as a novel binding partner of 53BP1. We find that TPX2/Aurora A plays a previously unrecognized role in DNA damage repair and replication fork stability by counteracting 53BP1 function. Loss of TPX2 or Aurora A compromises DNA end resection, BRCA1 and Rad51 recruitment, and homologous recombination. Furthermore, loss of TPX2 or Aurora A causes deprotection of stalled replication forks upon replication stress induction. This fork protection pathway counteracts MRE11 nuclease activity but functions in parallel to BRCA1. Strikingly, concurrent loss of 53BP1 rescues not only BRCA1/Rad51 recruitment but also the fork instability induced upon TPX2 loss. Our work suggests the presence of a feedback mechanism by which 53BP1 is regulated by a novel binding partner and uncovers a unique role for 53BP1 in replication fork stability.
TL;DR: This work systematically measure proteomic and phosphoproteomic responses to perturbations of phosphorylation signaling networks in yeast deletion strains using a streamlined tandem mass tag (SL-TMT) strategy to understand the roles of kinases and phosphatases from a protein homeostasis point of view.
TL;DR: The approach to statistically humanize the mouse networks with data from human cancer and identified genes within the humanized CRC and PDAC networks synthetically lethal with mutant KRAS to demonstrate the context-dependent plasticity of oncogenic signaling.
Abstract: The highest frequencies of KRAS mutations occur in colorectal carcinoma (CRC) and pancreatic ductal adenocarcinoma (PDAC). The ability to target downstream pathways mediating KRAS oncogenicity is limited by an incomplete understanding of the contextual cues modulating the signaling output of activated K-RAS. We performed mass spectrometry on mouse tissues expressing wild-type or mutant Kras to determine how tissue context and genetic background modulate oncogenic signaling. Mutant Kras dramatically altered the proteomes and phosphoproteomes of preneoplastic and neoplastic colons and pancreases in a context-specific manner. We developed an approach to statistically humanize the mouse networks with data from human cancer and identified genes within the humanized CRC and PDAC networks synthetically lethal with mutant KRAS. Our studies demonstrate the context-dependent plasticity of oncogenic signaling, identify non-canonical mediators of KRAS oncogenicity within the KRAS-regulated signaling network, and demonstrate how statistical integration of mouse and human datasets can reveal cross-species therapeutic insights.
TL;DR: A TKO Visualization Tool (TVT) is presented, allowing the proteomics community to search and compare TKO results to optimize user-specific data collection workflows and to measure degradation of instrument performance over time.
Abstract: Multiplexing strategies are at the forefront of mass-spectrometry-based proteomics, with SPS-MS3 methods becoming increasingly commonplace. A known caveat of isobaric multiplexing is interference resulting from coisolated and cofragmented ions that do not originate from the selected precursor of interest. The triple knockout (TKO) standard was designed to benchmark data collection strategies to minimize interference. However, a limitation to its widespread use has been the lack of an automated analysis platform. We present a TKO Visualization Tool (TVT). The TVT viewer allows for automated, web-based, database searching of the TKO standard, returning traditional figures of merit, such as peptide and protein counts, scan-specific ion accumulation times, as well as the TKO-specific metric, the IFI (interference-free index). Moreover, the TVT viewer allows for plotting of two TKO standards to assess protocol optimizations, compare instruments, or measure degradation of instrument performance over time. We sh...
TL;DR: A multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs) is developed and it is demonstrated that doxorubicin induces M HC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins.
Abstract: MHC-I peptides are intracellular-cleaved peptides, usually 8-11 amino acids in length, which are presented on the cell surface and facilitate CD8+ T cell responses. Despite the appreciation of CD8+ T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8+ T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8+ T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8+ T-cell-based immunotherapies.
TL;DR: A regulatory mechanism operating as a molecular switch is described, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.
TL;DR: A growth factor and nutrient-stimulated pathway, whereby mammalian target of rapamycin (mTOR) activates Per–Arnt–Sim domain kinase (PASK) protein kinase in muscle stem cells is described, showing that phosphorylation of PASK by mTORC1 is required for the activation of myogenin transcription, exit from self-renewal, and induction of the myogenesis program.
Abstract: During skeletal muscle regeneration, muscle stem cells (MuSCs) respond to multiple signaling inputs that converge onto mammalian target of rapamycin complex 1 (mTORC1) signaling pathways. mTOR function is essential for establishment of the differentiation-committed progenitors (early stage of differentiation, marked by the induction of myogenin expression), myotube fusion, and, ultimately, hypertrophy (later stage of differentiation). While a major mTORC1 substrate, p70S6K, is required for myotube fusion and hypertrophy, an mTORC1 effector for the induction of myogenin expression remains unclear. Here, we identified Per-Arnt-Sim domain kinase (PASK) as a downstream phosphorylation target of mTORC1 in MuSCs during differentiation. We have recently shown that the PASK phosphorylates Wdr5 to stimulate MuSC differentiation by epigenetically activating the myogenin promoter. We show that phosphorylation of PASK by mTORC1 is required for the activation of myogenin transcription, exit from self-renewal, and induction of the myogenesis program. Our studies reveal that mTORC1-PASK signaling is required for the rise of myogenin-positive committed myoblasts (early stage of myogenesis), whereas mTORC1-S6K signaling is required for myoblast fusion (later stage of myogenesis). Thus, our discoveries allow molecular dissection of mTOR functions during different stages of the myogenesis program driven by two different substrates.
TL;DR: Data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses and should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.
Abstract: Oncolytic 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.
TL;DR: This work shows that a subset of glucose transporters was among the most downregulated proteins in the cell upon arsenic exposure, and shows that this downregulation reflects targeted arsenic-dependent degradation of glucosetransporters.
Abstract: The abundance of cell surface glucose transporters must be precisely regulated to ensure optimal growth under constantly changing environmental conditions. We recently conducted a proteomic analysis of the cellular response to trivalent arsenic, a ubiquitous environmental toxin and carcinogen. A surprising finding was that a subset of glucose transporters was among the most downregulated proteins in the cell upon arsenic exposure. Here we show that this downregulation reflects targeted arsenic-dependent degradation of glucose transporters. Degradation occurs in the vacuole and requires the E2 ubiquitin ligase Ubc4, the E3 ubiquitin ligase Rsp5, and K63-linked ubiquitin chains. We used quantitative proteomic approaches to determine the ubiquitinated proteome after arsenic exposure, which helped us to identify the ubiquitination sites within these glucose transporters. A mutant lacking all seven major glucose transporters was highly resistant to arsenic, and expression of a degradation-resistant transporter restored arsenic sensitivity to this strain, suggesting that this pathway represents a protective cellular response. Previous work suggests that glucose transporters are major mediators of arsenic import, providing a potential rationale for this pathway. These results may have implications for the epidemiologic association between arsenic exposure and diabetes.
TL;DR: In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down, and it is shown that the lower band is likely non-specific.
Abstract: In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.
TL;DR: The TomahaqCompanion application is introduced, an open-source desktop application that enables rapid creation of TOMAHAQ methods and analysis of quantitative TOMaHAQ data and demonstrates the flexibility of TomahaquCompanion by creating a variety of methods testing TOMAhaQ parameters.
Abstract: Triggered by Offset, Multiplexed, Accurate mass, High resolution, and Absolute Quantitation (TOMAHAQ) is a recently introduced targeted proteomics method that combines peptide and sample multiplexing. TOMAHAQ assays enable sensitive and accurate multiplexed quantification by implementing an intricate data collection scheme that comprises multiple MSn scans, mass inclusion lists, and data-driven filters. Consequently, manual creation of TOMAHAQ methods can be time-consuming and error prone, while the resulting TOMAHAQ data may not be compatible with common mass spectrometry analysis pipelines. To address these concerns we introduce TomahaqCompanion, an open-source desktop application that enables rapid creation of TOMAHAQ methods and analysis of TOMAHAQ data. Starting from a list of peptide sequences, a user can perform each step of TOMAHAQ assay development including (1) generation of priming run target list, (2) analysis of priming run data, (3) generation of TOMAHAQ method file, and (4) analysis and export of quantitative TOMAHAQ data. We demonstrate the flexibility of TomahaqCompanion by creating a variety of methods testing TOMAHAQ parameters (e.g., number of SPS notches, run length, etc.). Lastly, we analyze an interference sample comprising heavy yeast peptides, a standard human peptide mixture, TMT11-plex, and super heavy TMT (shTMT) isobaric labels to demonstrate ∼10-200 attomol limit of quantification within a complex background using TOMAHAQ.
TL;DR: A new, real-time database search platform, Orbiter, is built to combat the SPS-MS3 method’s longer duty cycles, and it is found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency.
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 enables the elimination of 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 milliseconds. 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 was able to quantify more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 hours for RTS, 36 hours for SPS-MS3).
TL;DR: This work reports an alternative, precursor-based quantification method using non-isobaric TMT variants: TMT0 (TMTzero) and shTMT (super-heavy TMT), and concludes that mTMT is a suitable alternative for precursor- based protein quantification.
Abstract: Stable isotope labeling of peptides is the basis for numerous mass-spectrometry-based quantification strategies. Isobaric tagging and metabolic labeling, namely, tandem mass tagging (TMT) and SILAC...
TL;DR: A mode of Hog1 function is identified whereby Hog1 senses arsenic through a direct physical interaction that requires three conserved cysteine residues located adjacent to the catalytic loop, which provide a mechanistic basis for understanding how stress-activated kinases can sense distinct threats and perform highly specific adaptive responses.
Abstract: The yeast stress-activated protein kinase Hog1 is best known for its role in mediating the response to osmotic stress, but it is also activated by various mechanistically distinct environmental stressors, including heat shock, endoplasmic reticulum stress, and arsenic. In the osmotic stress response, the signal is sensed upstream and relayed to Hog1 through a kinase cascade. Here, we identified a mode of Hog1 function whereby Hog1 senses arsenic through a direct physical interaction that requires three conserved cysteine residues located adjacent to the catalytic loop. These residues were essential for Hog1-mediated protection against arsenic, were dispensable for the response to osmotic stress, and promoted the nuclear localization of Hog1 upon exposure of cells to arsenic. Hog1 promoted arsenic detoxification by stimulating phosphorylation of the transcription factor Yap8, promoting Yap8 nuclear localization, and stimulating the transcription of the only known Yap8 targets, ARR2 and ARR3, both of which encode proteins that promote arsenic efflux. The related human kinases ERK1 and ERK2 also bound to arsenic in vitro, suggesting that this may be a conserved feature of some members of the mitogen-activated protein kinase (MAPK) family. These data provide a mechanistic basis for understanding how stress-activated kinases can sense distinct threats and perform highly specific adaptive responses.
TL;DR: Monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by the small molecule SHP099 reveals six distinct response signatures following SHp099 treatment and washout, highlighting how analysis of phospho-abundance dynamics can provide insight into transmembrane signaling responses.
Abstract: SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines; it is frequently mutated in childhood leukemias and other cancers. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by the small molecule SHP099. Data on phosphotyrosine abundance at more than 400 tyrosine residues reveals six distinct response signatures following SHP099 treatment and washout. These include putative substrate sites with increased phosphotyrosine abundance at early or late timepoints, and another class of sites that shows reduced phosphotyrosine abundance when SHP099 is present. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight how analysis of phosphoproteomic dynamics can provide insight into transmembrane signaling responses.
TL;DR: The results suggest that budding yeast shares with other eukaryotes the ability to establish complex heterochromatin domains that coordinate multiple mechanisms of silencing regulation through physical interactions.
Abstract: Heterochromatin domains are stably repressed chromatin structures composed of a core assembly of silencing proteins that condense adjacent nucleosomes. The minimal heterochromatin structure can serve as a platform for recruitment of complementary regulatory factors. We find that a reconstituted budding yeast heterochromatin domain can act as a platform to recruit multiple factors that play a role in regulating heterochromatin function. We uncover the direct interaction between the SIR heterochromatin complex and a chromosomal boundary protein that restricts the spread of heterochromatin. We find that the SIR complex relieves a mechanism of auto-inhibition within the boundary protein Yta7, allowing the Yta7 bromodomain to engage chromatin. Our results suggest that budding yeast shares with other eukaryotes the ability to establish complex heterochromatin domains that coordinate multiple mechanisms of silencing regulation through physical interactions.
TL;DR: A novel, shape-independent method, named Analysis of Independent Differences (AID), which detects complex melting profiles and can be extended to any number of temperature channels, ligand-protein or protein-protein interactions, or general curve data for deeper biological insight.
Abstract: Identifying global cellular targets of small molecules is a challenge for drug discovery. Thermal proteome profiling (TPP) is a recent technique that uses quantitative proteomics to identify all small molecule protein targets in a single experiment. One current TPP analysis method relies on two major assumptions: sigmoidal melting curve behavior and that intra-condition dependencies preclude an independent and identically distributed model. Herein, we use a previously published panobinostat TPP dataset to show that these assumptions do not hold true and present a novel, shape-independent method, named Analysis of Independent Differences (AID). For each temperature, AID models the differences between conditions of fractions of non-denatured protein as an independent Normal distribution, resulting in a Multivariate Normal observation for each protein. The log of a Multivariate Normal p-value ranks the proteins from most to least likely shifted, and individual Normal p-values within each protein allow for qualitative inspection. Applying AID to the panobinostat dataset revealed known targets in the top 3% of most shifted proteins, as well as candidate targets involved in myeloid leukocyte activation. AID detects complex melting profiles and can be extended to any number of temperature channels, ligand-protein or protein-protein interactions, or general curve data for deeper biological insight.
TL;DR: A role is identified for the highly conserved and essential Rix1-containing complex, with known RNA endonuclease and polynucleotide kinase activities required for ribosomal RNA (rRNA) processing, in spreading and epigenetic inheritance of heterochromatin.
Abstract: Heterochromatin assembly requires the methylation of histone H3 lysine 9 by the Clr4(Suv39h) methyltransferase and both the spreading and epigenetic inheritance of heterochromatin involve the recognition of H3K9me-containing nucleosomes by Clr4 and catalysis of H3K9me on adjacent nucleosomes. How this read-write mechanism overcomes obstacles posed by RNA polymerase II and nascent RNA in its path is not fully understood. Here we identify a role for the highly conserved and essential Rix1-containing complex (here referred to as the rixosome), with known RNA endonuclease and polynucleotide kinase activities required for ribosomal RNA (rRNA) processing, in spreading and epigenetic inheritance of heterochromatin. Viable mutations in rixosome subunits 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 away from nucleation sites into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.
TL;DR: This manuscript was submitted in error therefore, the authors do not wish this work to be cited as reference for the project.
Abstract: SUMMARY Rapid alterations in cellular metabolism allow tissues to maintain homeostasis during changes in energy availability. Acetyl-CoA carboxylase 2 (ACC2) provides a central regulation and is phosphorylated acutely by AMPK during cellular stress to relieve repression of fat oxidation. While ACC2 is hydroxlated by prolyl hydroxylase 3 (PHD3), the physiological consequence of PHD3 is little understood. We find that ACC2 phosphorylation and hydroxylation occur in a reciprocal fashion. ACC2 hydroxylation occurs in conditions of high energy and leads to decreased fatty acid oxidation. Furthermore, AMPK-mediated phosphorylation of ACC2 is inhibitory to hydroxylation. PHD3 null mice demonstrate loss of ACC2 hydroxylation in heart and skeletal muscle and display elevated fatty acid oxidation. Whole body or skeletal muscle-specific PHD3 loss enhanced exercise capacity during an endurance exercise challenge. In sum, these data identify PHD3 as a physiological regulator of energy balance during acute stress.