We have previously identified the cytoplasmic prolyl tRNA synthetase in Plasmodium falciparum as the functional target of the natural product febrifugine and its synthetic analogue halofuginone (HFG), one of the most potent antimalarials discovered to date. However, our studies also discovered that short-term treatment of asexual blood stage P. falciparum with HFG analogues causes a 20-fold increase in intracellular proline, termed the adaptive proline response (APR), which renders parasites tolerant to HFG. This novel resistance phenotype lacks an apparent genetic basis but remains stable after drug withdrawal. On the basis of our findings that HFG treatment induces eIF2α phosphorylation, a sensitive marker and mediator of cellular stress, we here investigate if eIF2α-signaling is functionally linked to the APR. In our comparative studies using a parasite line lacking PfeIK1, the Plasmodium orthologue of the eIF2α-kinase GCN2 that mediates amino acid deprivation sensing, we show that HFG activity and the APR are independent from PfeIK1 and eIF2α signaling.
Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive β-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of β-catenin, define functional networks required for the survival of β-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.
Immunoaffinity enrichment of peptides coupled with analysis by stable isotope dilution multiple reaction mass spectrometry has been shown to have analytical performance and detection limits suitable for many biomarker verification studies and biological applications. Prior studies have shown that antipeptide antibodies can be multiplexed up to 50 in a single assay without significant loss of performance. Achieving higher multiplex levels is relevant to all studies involving precious biological material as this minimizes the amount of sample that must be consumed to measure a given set of analytes and reduces the assay cost per analyte. Here we developed automated methods employing the Agilent AssayMAP Bravo microchromatography platform and used these methods to characterize the performance of immunoaffinity enrichment of peptides up to multiplex levels of 172. Median capture efficiency for the target peptides remained high (88%) even at levels of 150-plex and declined to 70% at 172-plex compared to antibody performance observed at standard lower multiplex levels (n = 25). Subsequently, we developed and analytically characterized a multiplexed immuno-multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) assay (n = 110) and applied it to measure candidate protein biomarkers of cardiovascular disease in plasma of patients undergoing planned myocardial infarction. The median lower limit of detection of all peptides was 71.5 amol/μL (nM), and the coefficient of variation (CV) was less than 15% at the lower limit of quantification. The results demonstrate that high multiplexed immuno-MRM-MS assays are readily achievable using the optimized sample processing and peptide capture methods described here.
Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.
The polymorphism ATG16L1 T300A, associated with increased risk of Crohn's disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1(∗)300A risk allele. Clec12a(-/-) mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease.
A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1β production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1β production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.
Variability in the reproducibility of protein digestion is the major source of measurement variance in stable isotope dilution, multiple reaction monitoring mass spectrometry (SID-MRM-MS), and immuno-MRM-MS (SISCAPA) assays. This application note describes how the Agilent Bravo Automated Liquid Handling Platform was used for automated digestion in order to improve the reproducibility between experimental runs and reduce the labor intensity of this step when large numbers of samples are evaluated. A Bravo Platform at the Broad Institute was configured for automated reduction, alkylation, and proteolytic digestion of plasma from human patients and animal samples to complete this assessment. This application note compares the automated trypsin digestion of plasma in 96-well plates using the Bravo Platform to manual trypsin digestion in microcentrifuge tubes, demonstrating comparable analytical results between manual and automated methods. Automation on the Bravo Platform allows us to process up to 96 samples per day (as compared to 24 processed manually), enables parallel reagent addition, and improves lab productivity. A future report will detail our use of the Agilent Bravo in targeted immuno-MRM enrichment.