Protein aggregation increases during aging and is a pathological hallmark of many age-related diseases. Protein homeostasis (proteostasis) depends on a core network of factors directly influencing protein production, folding, trafficking, and degradation. Cellular proteostasis also depends on the overall composition of the proteome and numerous environmental variables.
View Article and Find Full Text PDFThe aminopeptidase, endoplasmic reticulum aminopeptidase 1 (ERAP1), trims peptides for loading into major histocompatibility complex class I (MHC class I), and loss of this activity has broad effects on the MHC class I peptidome. Here, we investigated the impact of targeting ERAP1 in immune checkpoint blockade (ICB), as MHC class I interactions mediate both activating and inhibitory functions in antitumor immunity. Loss of ERAP sensitized mouse tumor models to ICB, and this sensitivity depended on CD8 T cells and natural killer (NK) cells.
View Article and Find Full Text PDFBlood plasma is one of the most commonly analyzed and easily accessible biological samples. Here, we describe an automated liquid-liquid extraction platform that generates accurate, precise, and reproducible samples for metabolomic, lipidomic, and proteomic analyses from a single aliquot of plasma while minimizing hands-on time and avoiding contamination from plasticware. We applied mass spectrometry to examine the metabolome, lipidome, and proteome of 90 plasma samples to determine the effects of age, time of day, and a high-fat diet in mice.
View Article and Find Full Text PDFOne of the main hallmarks of aging is aging-associated inflammation, also known as inflammaging. In this study, by comparing plasma and kidney proteome profiling of young and old mice using LC-MS profiling, we discovered that immunoglobulins are the proteins that exhibit the highest increase with age. This observation seems to have been disregarded because conventional proteome profiling experiments typically overlook the expression of high-abundance proteins or employ depletion methods to remove them before LC-MS analysis.
View Article and Find Full Text PDFWe present a framework for the analysis of multiplexed mass spectrometry proteomics data that reduces estimation error when combining multiple isobaric batches. Variations in the number and quality of observations have long complicated the analysis of isobaric proteomics data. Here we show that the power to detect statistical associations is substantially improved by utilizing models that directly account for known sources of variation in the number and quality of observations that occur across batches.
View Article and Find Full Text PDFWe describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types.
View Article and Find Full Text PDFStochastic, intensity-based precursor isolation can result in isotopically enriched fragment ions. This problem is exacerbated for large peptides and stable isotope labeling experiments using deuterium or N. For stable isotope labeling experiments, incomplete and ubiquitous labeling strategies result in the isolation of peptide ions composed of many distinct structural isomers.
View Article and Find Full Text PDFSystemic lupus erythematosus is a common autoimmune inflammatory disease which is associated with increases in autoantibodies and immune complexes that deposit in the kidney. The MRL-lpr mouse is a common mouse model used for the study of lupus and immune complex glomerulonephritis but very little is known about the plasma proteome changes in this model. We performed in-depth quantitative proteome profiling on MRL-lpr and control (strain MpJ) mice to investigate the changes in the proteome, immunoglobulins and their glycoproteome as well as protein and immune complexes.
View Article and Find Full Text PDFThe process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq.
View Article and Find Full Text PDFPerforming large-scale plasma proteome profiling is challenging due to limitations imposed by lengthy preparation and instrument time. We present a fully automated multiplexed proteome profiling platform (AutoMP3) using the Hamilton Vantage liquid handling robot capable of preparing hundreds to thousands of samples. To maximize protein depth in single-shot runs, we combined 16-plex Tandem Mass Tags (TMTpro) with high-field asymmetric waveform ion mobility spectrometry (FAIMS Pro) and real-time search (RTS).
View Article and Find Full Text PDFOnly a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to identify perturbations that enhance natural killer effector functions. Our screens defined critical components of the tumor-immune synapse and highlighted the importance of cancer cell interferon-γ signaling in modulating NK activity.
View Article and Find Full Text PDFThe integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity.
View Article and Find Full Text PDFOne of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205.
View Article and Find Full Text PDFObesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ (peroxisome proliferator-activated receptor γ) at serine 273 by cyclin-dependent kinase 5 (Cdk5) stimulates diabetogenic gene expression in adipose tissues. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic drugs that bind PPARγ, such as the thiazolidinediones and PPARγ partial agonists or non-agonists.
View Article and Find Full Text PDFMultiplexed isobaric tag based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of mitogen-activated protein/extracellular signal-regulated kinase (MEK) signaling in cancer and mitogen-activated protein kinase (MAPK)-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of tandem mass tag 10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice.
View Article and Find Full Text PDFThe ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult.
View Article and Find Full Text PDFMutations in the acid β-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gaucher's disease (GD), are the strongest genetic risk factor for Parkinson's disease (PD) known to date. Here we generate induced pluripotent stem cells from subjects with GD and PD harbouring GBA1 mutations, and differentiate them into midbrain dopaminergic neurons followed by enrichment using fluorescence-activated cell sorting. Neurons show a reduction in glucocerebrosidase activity and protein levels, increase in glucosylceramide and α-synuclein levels as well as autophagic and lysosomal defects.
View Article and Find Full Text PDFCarboxyl-ester lipase (CEL) maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes and pancreatic exocrine dysfunction due to mutations in the CEL gene encoding CEL. The pathogenic mechanism for diabetes development is unknown. Since CEL is expressed mainly in pancreatic acinar cells, we asked whether we could find structural pancreatic changes in CEL-MODY subjects during the course of diabetes development.
View Article and Find Full Text PDFTargeted proteomics assays such as those measuring end points in activity assays are sensitive and specific but often lack in throughput. In an effort to significantly increase throughput, a comparison was made between the traditional approach which utilizes an internal standard and the multiplexing approach which relies on isobaric tagging. A kinase activity assay was used for proof of concept, and experiments included three biological replicates for every condition.
View Article and Find Full Text PDFBackground: CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas.
View Article and Find Full Text PDFArguably the most important issue in phosphorylation studies is the determination of kinase-substrate relationships. Kinase Activity-Abundance Correlation (KAAC) profiling is a technique that can be used to suggest the kinase responsible for a particular phosphorylation event. The method involves the separation of a lysate at the protein level, after which fractions are analyzed to produce an activity profile for each substrate peptide.
View Article and Find Full Text PDFDeamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.
View Article and Find Full Text PDFProtein phosphorylation is an important and ubiquitous post-translational modification in eukaryotic biological systems. The KAYAK (Kinase ActivitY Assay for Kinome profiling) assay measures the phosphorylation rates of dozens of peptide substrates simultaneously, directly from cell lysates. Here, we simplified the assay by removing the phosphopeptide enrichment step, increasing throughput while maintaining similar data quality.
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