Search

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

Advanced GIST is characterized by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor (CDK4/6i) therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximizing response to therapeutic CDK4/6 inhibition. Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples (N=18), including 8 metastatic TKI-resistant GISTs. Multiple metastases were analyzed in 3 patients. The impact of CDK2i (CDK2 inhibitor-II), CDK4/6i (palbociclib or abemaciclib), and CDK2/4/6i (PF-06873600) was determined through cell proliferation and protein detection assays in vitro and in vivo. Mechanisms of acquired CDK2i and CDK4/6i resistance were characterized in GIST cell lines after long-term exposure. The results demonstrate recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways. Identical aberrations of p16, RB1, and TP53 were present in all metastases from 3 patients. We show that therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation (P Citation Format: Inga-Marie Schaefer, Matthew L. Hemming, Meijun Z. Lundberg, Matthew P. Serrata, Isabel Goldaracena, Ninning Liu, Peng Yin, Joao A. Paulo, Steven P. Gygi, Suzanne George, Jeffrey A. Morgan, Monica M. Bertagnolli, Ewa T. Sicinska, Adrian Mariño-Enríquez, Jason L. Hornick, Chandrajit P. Raut, George D. Demetri, Wen-Bin Ou, Sinem K. Saka, Jonathan A. Fletcher. CDK2 and CDK4/6 inhibition in GIST: Mechanisms of response and resistance [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A013.

Insulin secretion from pancreatic β cells is essential for glucose homeostasis. An insufficient response to the demand for insulin results in diabetes. We previously showed that β cell-specific deletion of Zfp148 (β-Zfp148KO) improves glucose tolerance and insulin secretion in mice. Here, we performed Ca2+ imaging of islets from β‑Zfp148KO and control mice fed both a chow and a Western-style diet. β-Zfp148KO islets demonstrated improved sensitivity and sustained Ca2+ oscillations in response to elevated glucose levels. β-Zfp148KO islets also exhibited elevated sensitivity to amino acid-induced Ca2+ influx under low glucose conditions, suggesting enhanced mitochondrial phosphoenolpyruvate-dependent (PEP-dependent), ATP-sensitive K+ channel closure, independent of glycolysis. RNA-Seq and proteomics of β-Zfp148KO islets revealed altered levels of enzymes involved in amino acid metabolism (specifically, SLC3A2, SLC7A8, GLS, GLS2, PSPH, PHGDH, and PSAT1) and intermediary metabolism (namely, GOT1 and PCK2), consistent with altered PEP cycling. In agreement with this, β-Zfp148KO islets displayed enhanced insulin secretion in response to l-glutamine and activation of glutamate dehydrogenase. Understanding pathways controlled by ZFP148 may provide promising strategies for improving β cell function that are robust to the metabolic challenge imposed by a Western diet.

Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We find that microbial metabolites from healthy mice or humans are growth-repressive, and this response is attenuated in mice and patients with CRC. Microbial profiling reveals that Lactobacillus reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC. Reuterin alters redox balance, and reduces proliferation and survival in colon cancer cells. Reuterin induces selective protein oxidation and inhibits ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restricts colon tumor growth, increases tumor reactive oxygen species, and decreases protein translation in vivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.

Zellweger spectrum disorder (ZSD) is the most severe peroxisomal biogenesis disorder (PBD). Why ZSD patients not only loose functional peroxisomes but also present with severe mitochondrial dysfunction was a long‐standing mystery. In this issue, Nuebel et al (2021) identified that loss of peroxisomes leads to re‐routing of peroxisomal proteins to mitochondria, thereby impairing mitochondrial structure and function. The findings provide the first molecular understanding of the mitochondrial‐peroxisomal link in ZSD., A study in this issue provides the first molecular understanding of the mitochondrial‐peroxisomal link in peroxisomal disorders.

Recent advances in mass spectrometry (MS) have enabled quantitative proteomics to become a powerful tool in the field of drug discovery, especially when applied toward proteome-wide target engagement studies. Similar to temperature gradients, increasing concentrations of organic solvents stimulate unfolding and precipitation of the cellular proteome. This property can be influenced by physical association with ligands and other molecules, making individual proteins more or less susceptible to solvent-induced denaturation. Herein, we report the development of proteome-wide solvent shift assays by combining the principles of solvent-induced precipitation (Zhang et al., 2020) with modern quantitative proteomics. Using this approach, we developed solvent proteome profiling (SPP), which is capable of establishing target engagement through analysis of SPP denaturation curves. We readily identified the specific targets of compounds with known mechanisms of action. As a further efficiency boost, we applied the concept of area-under-the-curve analysis to develop solvent proteome integral solubility alteration (solvent-PISA) and demonstrate that this approach can serve as a reliable surrogate for SPP. We propose that by combining SPP with alternative methods, like thermal proteome profiling, it will be possible to increase the absolute number of high-quality melting curves that are attainable by either approach individually thereby increasing the fraction of the proteome that can be screened for evidence of ligand binding.

Wnt5a-Ror signaling is a conserved pathway that regulates morphogenetic processes during vertebrate development [R. T. Moon et al., Development 119, 97–111 (1993); I. Oishi et al., Genes Cells 8, 645–654 (2003)], but its downstream signaling events remain poorly understood. Through a large-scale proteomic screen in mouse embryonic fibroblasts, we identified the E3 ubiquitin ligase Pdzrn3 as a regulatory target of the Wnt5a-Ror pathway. Upon pathway activation, Pdzrn3 is degraded in a β-catenin–independent, ubiquitin-proteasome system–dependent manner. We developed a flow cytometry-based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 stability. Epistatically, Pdzrn3 is regulated independently of Kif26b, another Wnt5a-Ror effector. Wnt5a-dependent degradation of Pdzrn3 requires phosphorylation of three conserved amino acids within its C-terminal LNX3H domain [M. Flynn, O. Saha, P. Young, BMC Evol. Biol. 11, 235 (2011)], which acts as a bona fide Wnt5a-responsive element. Importantly, this phospho-dependent degradation is essential for Wnt5a-Ror modulation of cell migration. Collectively, this work establishes a Wnt5a-Ror cell morphogenetic cascade involving Pdzrn3 phosphorylation and degradation.

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.

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, are among the most widely used techniques for relative protein quantification. Here we report an alternative, precursor-based quantification method using nonisobaric TMT variants: TMTzero (TMT0) and superheavy TMT (shTMT). We term this strategy mass difference tandem mass tagging (mTMT). These TMT variants differ by 11 mass units; however, peptides labeled with these reagents coelute, analogous to SILAC-labeled peptide pairs. As a proof-of-concept, we profiled the proteomes of two cell lines that are frequently used in neuroscience studies, SH-SY5Y and SVGp12, using mTMT and standard SILAC-labeling approaches. We show similar quantified proteins and peptides for each method, with highly correlated fold-changes between workflows. We conclude that mTMT is a suitable alternative for precursor-based protein quantification.

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.

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 MS(2) (HRMS(2)) and synchronous precursor selection MS(3) (SPS-MS(3))-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.

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-MS(3) 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-MS(3) method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS(3) approach provides dramatic performance improvements for quantitative multiplexed experiments.

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 MS(n) 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.

Significance T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. Finding new ways to boost T cell immunity in the elderly is key for enhancing their immune competence. In this work, we performed a systematic analysis of proteins and metabolites in young versus aged T cells. Metabolic rewiring occurs in young T cells following stimulation but is dampened in aged T cells. Moreover, we show that aged T cell functions can be enhanced by metabolite addition., T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. While cellular metabolism tightly regulates T cell activation and function, metabolic reprogramming in aged T cells has not been thoroughly studied. Here, we report a systematic analysis of metabolism during young versus aged naïve T cell activation. We observed a decrease in the number and activation of naïve T cells isolated from aged mice. While young T cells demonstrated robust mitochondrial biogenesis and respiration upon activation, aged T cells generated smaller mitochondria with lower respiratory capacity. Using quantitative proteomics, we defined the aged T cell proteome and discovered a specific deficit in the induction of enzymes of one-carbon metabolism. The activation of aged naïve T cells was enhanced by addition of products of one-carbon metabolism (formate and glycine). These studies define mechanisms of skewed metabolic remodeling in aged T cells and provide evidence that modulation of metabolism has the potential to promote immune function in aged individuals.

Targeted mass spectrometry-based assays typically rely on previously acquired large data sets for peptide target selection. Such repositories are widely available for unlabeled peptides. However, they are less common for isobaric tagged peptides. Here we have assembled two series of six data sets originating from a mouse embryonic fibroblast cell line (NIH/3T3). One series is of peptides derived from a tryptic digest of a whole cell proteome and a second from enriched phosphopeptides. These data sets encompass three labeling approaches (unlabeled, TMT11-labeled, and TMTpro16-labeled) and two data acquisition strategies (ion trap MS2 with and without FAIMS-based gas phase separation). We identified a total of 1 509 526 peptide-spectrum matches which covered 11 482 proteins from the whole cell proteome tryptic digest, and 188 849 phosphopeptides from the phosphopeptide enrichment. The data sets were of similar depth, and while overlap across data sets was modest, protein overlap was high, thus reinforcing the comprehensiveness of these data sets. The data also supported FAIMS as a means to increase data set depth. These data sets provide a rich resource of peptides that may be used as starting points for targeted assays. Future data sets may be compiled for any genome-sequenced organism using the technologies and strategies highlighted herein. The data have been deposited in the ProteomeXchange Consortium with data set identifier PXD024298.

EGFR endocytosis is triggered by ligand binding or activation of p38 MAPK. Perez Verdaguer et al. elucidate phosphorylation-dependent mechanisms of p38-induced endocytosis of EGFR and dissect the interplay between ligand- and p38-induced clathrin-mediated and clathrin-independent pathways of EGFR endocytosis., Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.

The development of streamlined and high-throughput sample processing workflows is important for capitalizing on emerging advances and innovations in mass spectrometry-based applications. While the adaptation of new technologies and improved methodologies is fast paced, automation of upstream sample processing often lags. Here we have developed and implemented a semiautomated paramagnetic bead-based platform for isobaric tag sample preparation. We benchmarked the robot-assisted platform by comparing the protein abundance profiles of six common parental laboratory yeast strains in triplicate TMTpro16-plex experiments against an identical set of experiments in which the samples were manually processed. Both sets of experiments quantified similar numbers of proteins and peptides with good reproducibility. Using these data, we constructed an interactive website to explore the proteome profiles of six yeast strains. We also provide the community with open-source templates for automating routine proteomics workflows on an opentrons OT-2 liquid handler. The robot-assisted platform offers a versatile and affordable option for reproducible sample processing for a wide range of protein profiling applications.

May et al. combine proximity labeling with quantitative mass spectrometry to profile the proteome of primary cilia in cells responding to Hedgehog. They identify signaling factors that undergo dynamic ciliary enrichment and uncover an unanticipated regulation of ciliary cAMP signaling., The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein–coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type–specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.

Nicotine is a prominent active compound in tobacco and many smoking cessation products. Some of the biological effects of nicotine are well documented in in vitro and in vivo systems; however, data are scarce concerning the time-dependent changes on protein and phosphorylation events in response to nicotine. Here, we profiled the proteomes of SH-SY5Y and A549 cell lines subjected to acute (15 min, 1 h and 4 h) or chronic (24 h, 48 h) nicotine exposures. We used sample multiplexing (TMTpro16) and quantified more than 9000 proteins and over 7000 phosphorylation events per cell line. Among our findings, we determined a decrease in mitochondrial protein abundance for SH-SY5Y, while we detected alterations in several immune pathways, such as the complement system, for A549 following nicotine treatment. We also explored the proposed association between smoking (specifically nicotine) and SARS-CoV2. Here, we found several host proteins known to interact with viral proteins that were affected by nicotine in a time dependent manner. This dataset can be mined further to investigate the potential role of nicotine in different biological contexts. SIGNIFICANCE: Smoking is a major public health issue that is associated with several serious chronic, yet preventable diseases, including stroke, heart disease, type 2 diabetes, cancer, and susceptibility to infection. Tobacco smoke is a complex mixture of thousands of different compounds, among which nicotine is the main addictive compound. The biological effects of nicotine have been reported in several models, however very little data are available concerning the temporal proteomic and phosphoproteomic changes in response to nicotine. Here, we provide a dataset exploring the potential role of nicotine on different biological processes over time, including implications in the study of SARS-CoV2.

Investigation of the molecular mechanisms of aging in the human heart is challenging because of confounding factors, such as diet and medications, as well as limited access to tissues from healthy aging individuals. The laboratory mouse provides an ideal model to study aging in healthy individuals in a controlled environment. However, previous mouse studies have examined only a narrow range of the genetic variation that shapes individual differences during aging. Here, we analyze transcriptome and proteome data from 185 genetically diverse male and female mice at ages 6, 12, and 18 mo to characterize molecular changes that occur in the aging heart. Transcripts and proteins reveal activation of pathways related to exocytosis and cellular transport with age, whereas processes involved in protein folding decrease with age. Additional changes are apparent only in the protein data including reduced fatty acid oxidation and increased autophagy. For proteins that form complexes, we see a decline in correlation between their component subunits with age, suggesting age-related loss of stoichiometry. The most affected complexes are themselves involved in protein homeostasis, which potentially contributes to a cycle of progressive breakdown in protein quality control with age. Our findings highlight the important role of post-transcriptional regulation in aging. In addition, we identify genetic loci that modulate age-related changes in protein homeostasis, suggesting that genetic variation can alter the molecular aging process.

Significance Understanding the functions of the genes encoded in the SARS-CoV-2 genome is imperative to understanding its pathogenesis. One unique feature of the SARS-CoV-2 genome is ORF10, a small putative protein that was hypothesized to promote infection by hijacking a cellular E3 ubiquitin ligase, CRL2ZYG11B. Here, we investigate whether ORF10 hijacks CRL2ZYG11B or functions in other ways, such as to inhibit CRL2ZYG11B or be degraded by it. We do not find evidence that ORF10 regulates or is regulated by CRL2ZYG11B, and, furthermore, we find that ZYG11B and its paralog are dispensable for SARS-CoV-2 infection in cultured cells., In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to “hijack” CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B. While we confirm the ORF10−ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B. Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. 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 SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. 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 the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.

Isobaric tag-based sample multiplexing strategies are extensively used for global protein abundance profiling. However, such analyses are often confounded by ratio compression resulting from the co-isolation, co-fragmentation, and co-quantification of co-eluting peptides, termed “interference.” Recent analytical strategies incorporating ion mobility and real-time database searching have helped to alleviate interference, yet further assessment is needed. Here, we present the strain-specific peptide (SSP) interference reference sample, a tandem mass tag (TMT)-pro-labeled quality control that leverages the genetic variation in the proteomes of eight phylogenetically divergent mouse strains. Typically, a peptide with a missense mutation has a different mass and retention time than the reference or native peptide. TMT reporter ion signal for the native peptide in strains that encode the mutant peptide suggests interference which can be quantified and assessed using the interference-free index (IFI). We introduce the SSP by investigating interference in three common data acquisition methods and by showcasing improvements in the IFI when using ion mobility-based gas-phase fractionation. In addition, we provide a user-friendly, online viewer to visualize the data and streamline the calculation of the IFI. The SSP will aid in developing and optimizing isobaric tag-based experiments.

Stable isotope labeling is a leading strategy for mass-spectrometry-based peptide quantification. Whereas TMTpro isobaric tagging can quantify up to 16 multiplexed samples in a single experiment, nonisobaric, yet chromatographically indistinguishable, variants of TMTpro reagents can be used in conjunction with the isobaric tag series for various peptide-targeting applications. Here we test the performance of two nonisobaric TMTpro variants, a stable-isotope-free TMTproZero tag and a nearly fully isotope-labeled "super-heavy" variant, shTMTpro, in a targeted assay for peptides of charge state 4+. We label each peptide with TMTproZero or Super Heavy TMTpro reagents and separately spike each peptide into a TMTpro16-labeled background (equal amount of peptide across all 16 channels). We observe that the expected 1:1 reporter ion ratio is distorted when a TMTproZero-labeled peptide is used; however, we note no such interference when shTMTpro substitutes the TMTproZero tag. Our data suggest that using the Super Heavy TMTpro reagent is an improvement over the TMTproZero reagent for the accurate quantification of high-charge-state peptides for trigger-based multiplexed assays.

The kidney is an excellent model for studying organ aging. Kidney function shows steady decline with age and is easy to assay using urine or blood samples. However, little is known about the molecular changes that take place in the kidney during the aging process. In order to better understand the molecular changes that occur with age, we measured mRNA and protein levels in 188 genetically diverse mice at ages 6, 12, and 18 months. We observed distinctive change in mRNA and protein levels as a function of age. Changes in both mRNA and protein are associated with increased immune infiltration and decreases in mitochondrial function. Proteins show a greater extent of change and reveal changes in a wide array of biological processes including unique, organ-specific features of aging in kidney. Most importantly, we observed functionally important age-related changes in protein that occur in the absence of corresponding changes in mRNA. Our findings suggest that mRNA profiling alone provides an incomplete picture of molecular aging in the kidney and that examination of changes in proteins is essential to understand aging processes that are not transcriptionally regulated.

Summary The target of Rapamycin complex1 (TORC1) senses and integrates several environmental signals, including amino acid (AA) availability, to regulate cell growth. Folliculin (FLCN) is a tumor suppressor (TS) protein in renal cell carcinoma, which paradoxically activates TORC1 in response to AA supplementation. Few tractable systems for modeling FLCN as a TS are available. Here, we characterize the FLCN-containing complex in Schizosaccharomyces pombe (called BFC) and show that BFC augments TORC1 repression and activation in response to AA starvation and supplementation, respectively. BFC co-immunoprecipitates V-ATPase, a TORC1 modulator, and regulates its activity in an AA-dependent manner. BFC genetic and proteomic networks identify the conserved peptide transmembrane transporter Ptr2 and the phosphoribosylformylglycinamidine synthase Ade3 as new AA-dependent regulators of TORC1. Overall, these data ascribe an additional repressive function to Folliculin in TORC1 regulation and reveal S. pombe as an excellent system for modeling the AA-dependent, FLCN-mediated repression of TORC1 in eukaryotes., Graphical abstract, Highlights • The S.pombe Folliculin complex, Bhd1-Fnp1 Complex (BFC) augments repression or activation of TORC1 in response to amino acid levels, similar to a rheostat • BFC interacts and regulates V-ATPase activity in response to amino acid levels • Proteomic data reveal Ptr2 and Ade3 as novel amino acid-dependent regulators of TORC1 • S.pombe is an excellent model for studying the tumor suppressor function of BFC and related proteins, Cellular physiology; Cell biology; Functional aspects of cell biology

Protein phosphorylation has long been recognized as an essential regulator of protein activity, structure, complex formation, and subcellular localization among other cellular mechanisms. However, interpretation of the changes in protein phosphorylation is difficult. To address this difficulty, we measured protein and phosphorylation site changes across 11 points of a time course and developed a method for categorizing phosphorylation site behavior relative to protein level changes using the diauxic shift in yeast as a model and TMT11 sample multiplexing. We classified quantified proteins into behavioral categories that reflected differences in kinase activity, protein complex structure, and growth and metabolic pathway regulation across different phases of the diauxic shift. These data also provide a valuable resource for the study of fermentative versus respiratory growth and set a new benchmark for temporal quantitative proteomics and phosphoproteomics for the diauxic shift in Saccharomyces cerevisiae. Data are available via ProteomeXchange with identifier PXD022741.

While cancer is an age-related disease, most studies focus on genetically engineered younger mouse models. Here, we uncover how cancer develops as a consequence of the naturally aged immune system in mice. B-cell lymphoma frequently occurs in aged mice and is associated with increased cell size, splenomegaly, and a novel clonal B-cell population. Age-emergent B cells clonally expand outside of germinal centers driven by somatic mutations, activated c-Myc and hypermethylated promoters, and both genetically and epigenetically recapitulate human follicular and diffuse-large B-cell lymphomas. Mechanistically, mouse cancerous B cells originate from age-associated B cells, which are atypical memory B cells. Age-associated B cells secrete a spectrum of proinflammatory cytokines and activate paracrinally the expression of c-Myc in surrounding B cells. Although clonal B cells are a product of an aging microenvironment, they evolve being self-sufficient and support malignancy when transferred into young mice. Inhibition of mTOR and c-Myc attenuates premalignant changes in B cells during aging and emerges as a therapeutic strategy to delay the onset of age-related lymphoma. Together, we uncover how aging generates cancerous B cells, characterize a model that captures the origin of spontaneous cancer during aging and identify interventions that may postpone age-associated lymphoma.

A new positive selection assay could enhance the discovery of compounds that degrade previously undruggable proteins., Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal (“down”) assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal (“up”) assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9–based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

Poison frogs sequester their chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about poison frog physiological adaptations that confer this unusual ability to bioaccumulate dietary alkaloids from the intestines to skin glands for storage. We conducted an alkaloid-feeding experiment with the Diablito poison frog (Oophaga sylvatica) to determine how quickly toxins are accumulated and how toxins impact frog physiology using quantitative proteomics. Diablito frogs were able to rapidly accumulate the alkaloid decahydroquinoline to the skin storage glands within four days of dietary exposure, with decahydroquinoline also detected in the intestines and liver. Alkaloid exposure impacted tissue physiology, with protein abundance shifting in the intestines, liver, and skin. Many proteins that increased in abundance with toxin accumulation are plasma glycoproteins, including components of the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with toxin accumulation are membrane proteins involved in small molecule transport and metabolism, including ABC transporters, solute carrier proteins, and cytochrome P450s. Overall, this work shows that poison frogs can rapidly accumulate alkaloid toxins, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues. More broadly, this study suggests that acute changes in diet may quickly change the chemical arsenal and physiology of poison frogs, which may have important consequences in their defense against predation.

The final stages of mammalian erythropoiesis involve enucleation, membrane and proteome remodeling, and organelle clearance. Concomitantly, the erythroid membrane skeleton establishes a unique pseudohexagonal spectrin meshwork that is connected to the membrane through junctional complexes. The mechanism and signaling pathways involved in the coordination of these processes are unclear. The results of our study revealed an unexpected role of the membrane skeleton in the modulation of proteome remodeling and organelle clearance during the final stages of erythropoiesis. We found that diaphanous-related formin mDia2 is a master regulator of the integrity of the membrane skeleton through polymerization of actin protofilament in the junctional complex. The mDia2-deficient terminal erythroid cell contained a disorganized and rigid membrane skeleton that was ineffective in detaching the extruded nucleus. In addition, the disrupted skeleton failed to activate the endosomal sorting complex required for transport-III (ESCRT-III) complex, which led to a global defect in proteome remodeling, endolysosomal trafficking, and autophagic organelle clearance. Chmp5, a component of the ESCRT-III complex, is regulated by mDia2-dependent activation of the serum response factor and is essential for membrane remodeling and autophagosome-lysosome fusion. Mice with loss of Chmp5 in hematopoietic cells in vivo resembled the phenotypes in mDia2-knockout mice. Furthermore, overexpression of Chmp5 in mDia2-deficient hematopoietic stem and progenitor cells significantly restored terminal erythropoiesis in vivo. These findings reveal a formin-regulated signaling pathway that connects the membrane skeleton to proteome remodeling, enucleation, and organelle clearance during terminal erythropoiesis.

Summary Resistance to platinum compounds is a major determinant of patient survival in high-grade serous ovarian cancer (HGSOC). To understand mechanisms of platinum resistance and identify potential therapeutic targets in resistant HGSOC, we generated a data resource composed of dynamic (±carboplatin) protein, post-translational modification, and RNA sequencing (RNA-seq) profiles from intra-patient cell line pairs derived from 3 HGSOC patients before and after acquiring platinum resistance. These profiles reveal extensive responses to carboplatin that differ between sensitive and resistant cells. Higher fatty acid oxidation (FAO) pathway expression is associated with platinum resistance, and both pharmacologic inhibition and CRISPR knockout of carnitine palmitoyltransferase 1A (CPT1A), which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. The results are further validated in patient-derived xenograft models, indicating that CPT1A is a candidate therapeutic target to overcome platinum resistance. All multiomic data can be queried via an intuitive gene-query user interface (https://sites.google.com/view/ptrc-cell-line)., Graphical abstract, Highlights • Multi-omic profiles of platinum-resistant and sensitive ovarian cancer models • Significant alterations in multiomic profiles after carboplatin exposure • Oxidative phosphorylation and fatty acid oxidation (FAO) implicated in resistance • FAO/CPT1A may be a candidate druggable pathway to overcome platinum resistance, Huang et al. report extensive multiomic profiling of preclinical models of high-grade serous ovarian cancer and identify molecular features associated with resistance to standard-of-care, platinum-based chemotherapy. Functional data are presented, demonstrating that CPT1A is a candidate therapeutic target to overcome platinum resistance.

The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III2+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. By-passing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depend on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly, which are necessary for optimal cell growth in conditions of nucleotide limitation. A chemical screen identifies DHODH inhibitors as robust activators of mitochondrial respirasome assembly. Lipidomics reveal that peroxisomal-derived ether phospholipids accumulate in mitochondria during nucleotide deprivation to drive proliferation.

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and β-adrenergic receptor ADRB2, are strongly (∼20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified ∼160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.

Isobaric tagging is a powerful strategy for global proteome profiling. A caveat of isobaric-tag-based quantification is "interference", which may be caused by coeluting peptides that are coisolated, cofragmented, and coanalyzed, thereby confounding quantitative accuracy. Here, we present a two-proteome standard that challenges the mass spectrometer to measure a range of protein abundance ratios in a background of potential interference. The HYpro16 standard consists of tandem mass tag (TMT) pro16-labeled human peptides at a 1:1 ratio across all channels into which is spiked TMTpro16-labeled yeast peptides in triplicate at 20:1, 10:1, 4:1, and 2:1 ratios. We showcase the HYpro16 standard by (1) altering the MS2 isolation window width and (2) examining different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3). Our data illustrate that wider isolation widths moderately increase the TMT signal, the benefits of which are offset by decreased ratio accuracy. We also show that using real-time database searching (RTS)-MS3 resulted in the most accurate ratios. Additionally, the number of quantified yeast proteins using RTS-MS3 approaches that of hrMS2 when using a yeast-specific database for real-time searching. In short, this quality control standard allows for the assessment of multiple quantitative measurements within a single run, which can be compared across instruments to benchmark and track performance.

MotivationDirected gene/protein regulatory networks inferred by applying directed acyclic graph (DAG) models to proteogenomic data has been shown effective for detecting causal biomarkers of clinical outcomes. However, there remain unsolved challenges in DAG learning to jointly model clinical outcome variables, which often take binary values, and biomarker measurements, which usually are continuous variables. Therefore, in this paper, we propose a new tool, DAGBagM, to learn DAGs with both continuous and binary nodes. By using appropriate models for continuous and binary variables, DAGBagM allows for either type of nodes to be parents or children nodes in the learned graph. DAGBagM also employs a bootstrap aggregating strategy to reduce false positives and achieve better estimation accuracy. Moreover, the aggregation procedure provides a flexible framework to robustly incorporate prior information on edges for DAG reconstruction.ResultsAs shown by simulation studies, DAGBagM performs better in identifying edges between continuous and binary nodes, as compared to commonly used strategies of either treating binary variables as continuous or discretizing continuous variables. Moreover, DAGBagM outperforms several popular DAG structure learning algorithms including the score-based hill climbing (HC) algorithm, constraint-based PC-algorithm (PC-alg), and the hybrid method max-min hill climbing (MMHC) even for constructing DAG with only continuous nodes. The HC implementation in the R package DAGBagM is much faster than that in a widely used DAG learning R package bnlearn. When applying DAGBagM to proteomics datasets from ovarian cancer studies, we identify potential prognostic protein biomarkers in ovarian cancer.Availability and implementationDAGBagMis made available as a github repositoryhttps://github.com/jie108/dagbagM.

The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key β-cell transcription factors necessary for β-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance β-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain β-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key β-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature β-cell gene expression profile.

Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

Peroxisomal Biogenesis Disorders (PBDs) are a class of inherited metabolic disorders with profound neurological and other phenotypes. The most severe PBDs are caused by mutations in peroxin genes, which result in nonfunctional peroxisomes typically through impaired protein import. In order to better understand the molecular causes of Zellweger Spectrum Disease (ZSD) -the most severe PBDs -, we investigated the fate of peroxisomal mRNAs and proteins in ZSD model systems. We found that loss of peroxisomal import has no effect on peroxin mRNA expression or translational efficiency. Instead, peroxin proteins—still produced at high levels— aberrantly accumulate on the mitochondrial membrane, impairing respiration and ATP generation. Finally, we rescued mitochondrial function in fibroblasts derived from human patients with ZSD by overexpressing ATAD1, an AAA-ATPase that functions in mitochondrial quality control. These findings might provide a new focus of PBD therapies in supporting quality control pathways that protect mitochondrial function.

Proteins constitute much of the structure and functional machinery of cells, forming signaling networks, metabolic pathways, and large multi-component complexes. Protein abundance is regulated at multiple levels spanning transcription, translation, recycling, and degradation to maintain proper balance and optimal function. To better understand how protein abundances are maintained across varying genetic backgrounds, we analyzed liver proteomes of three genetically diverse mouse populations. We observe strong concordance of genetic and sex effects across populations. Differences between the populations arise from the contributions of additive, dominance, and epistatic components of heritable variation. We find that the influence of genetic variation on proteins that form complexes relates to their co-abundance. We identify effects on protein abundance from mutations that arose and became fixed during breeding and can lead to unique regulatory responses and disease states. Genetically diverse mouse populations provide powerful tools for understanding proteome regulation and its relationship to whole-organism phenotypes.