Tumor-specific mutations can generate neoantigens that drive CD8 T cell responses against cancer. Next-generation sequencing and computational methods have been successfully applied to identify ...mutations and predict neoantigens. However, only a small fraction of predicted neoantigens are immunogenic. Currently, predicted peptide binding affinity for MHC-I is often the major criterion for prioritizing neoantigens, although little progress has been made toward understanding the precise functional relationship between affinity and immunogenicity. We therefore systematically assessed the immunogenicity of peptides containing single amino acid mutations in mouse tumor models and divided them into two classes of immunogenic mutations. The first comprises mutations at a nonanchor residue, for which we find that the predicted absolute binding affinity is predictive of immunogenicity. The second involves mutations at an anchor residue; here, predicted relative affinity (compared with the WT counterpart) is a better predictor. Incorporating these features into an immunogenicity model significantly improves neoantigen ranking. Importantly, these properties of neoantigens are also predictive in human datasets, suggesting that they can be used to prioritize neoantigens for individualized neoantigen-specific immunotherapies.
Histone posttranslational modification leads to downstream effects indirectly by allowing or preventing docking of effector molecules, or directly by changing the intrinsic biophysical properties of ...local chromatin. To date, little has been done to study posttranslational modifications that lie outside of the unstructured tail domains of histones. Core residues, and in particular arginines in H3 and H4, mediate key interactions between the histone octamer and DNA in forming the nucleosomal particle. Using mass spectrometry, we find that one of these core residues, arginine 42 of histone H3 (H3R42), is dimethylated in mammalian cells by the methyltransferases coactivator arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 6 (PRMT6) in vitro and in vivo, and we demonstrate that methylation of H3R42 stimulates transcription in vitro from chromatinized templates. Thus, H3R42 is a new, “nontail” histone methylation site with positive effects on transcription. We propose that methylation of basic histone residues at the DNA interface may disrupt histone:DNA interactions, with effects on downstream processes, notably transcription.
Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial ...fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.
Identifying the protein targets of bioactive small molecules remains a major problem in the discovery of new chemical probes and therapeutics. While activity-based probes and photo-cross-linkers have ...had success in identifying protein targets of small molecules, each technique has limitations. Here we describe a method for direct proximity tagging of proteins that bind small molecules. We engineered a promiscuous ligase based on the NEDD8 conjugating enzyme, Ubc12, which can be covalently linked to a small molecule of interest. When target proteins bind the small molecule, they are directly labeled on surface lysines with a biotinylated derivative of the small ubiquitin homologue, NEDD8. This unique covalent tag can then be used to identify the small molecule binding proteins. Utilizing the drug dasatinib, we have shown that dasatinib-directed NEDDylation occurs for known endogenous protein binders in complex cell lysates. In addition, we have been able to improve NEDDylation efficiency through rational mutagenesis. Finally, we have shown that affinity-directed NEDDylation can be applied to two other protein–ligand interactions beyond kinases. Proximity tagging using this engineered ligase requires direct binding of the target and, thus, provides a useful and orthogonal approach to facilitate small molecule target identification.
Advances in several key technologies, including MHC peptidomics, have helped fuel our understanding of basic immune regulatory mechanisms and the identification of T cell receptor targets for the ...development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput and reproducibility would enable rapid characterization of multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, can be enriched in a semiautomated fashion on reusable, dry-storage, customized antibody cartridges. Using this method, a researcher, with very little hands-on time and in a single day, can perform up to 96 simultaneous enrichments at a similar level of quality as a manual workflow. TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification), a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. This unique combination of robust semiautomated MHC-I peptide isolation and high-throughput multiplexed targeted quantitation allows for both the routine analysis of >4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS.
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•Semiautomated peptide immunoprecipitation on reusable antibody cartridges.•Application of TOMAHAQ for MHC-I detection and quantitation.•Routine analysis of >4000 unique MHC-I peptides from 250 million cells via automation.•Quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS.
This manuscript highlights a new semiautomated MHC-I peptide immunoprecipitation method used in conjunction with the multiplexed quantitative technique TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification) for the detection and quantitation of MHC-I peptides. This combination of techniques allows the routine analysis of >4000 unique MHC-I peptides from 250 million cells and in with targeted analysis, quantitative sensitivity down to the low amol/μl level.
The low reactivity of peptide-prolyl-thioesters in native chemical ligation is not due to steric effects at the β-carbon, but rather to the presence of a carbonyl moiety on the nitrogen atom of the ...proline.
Development of tool molecules that inhibit Jumonji demethylases allows for the investigation of cancer-associated transcription. While scaffolds such as 2,4-pyridinedicarboxylic acid (2,4-PDCA) are ...potent inhibitors, they exhibit limited selectivity. To discover new inhibitors for the KDM4 demethylases, enzymes overexpressed in several cancers, we docked a library of 600 000 fragments into the high-resolution structure of KDM4A. Among the most interesting chemotypes were the 5-aminosalicylates, which docked in two distinct but overlapping orientations. Docking poses informed the design of covalently linked fragment compounds, which were further derivatized. This combined approach improved affinity by ∼3 log-orders to yield compound 35 (K i = 43 nM). Several hybrid inhibitors were selective for KDM4C over the related enzymes FIH, KDM2A, and KDM6B while lacking selectivity against the KDM3 and KDM5 subfamilies. Cocrystal structures corroborated the docking predictions. This study extends the use of structure-based docking from fragment discovery to fragment linking optimization, yielding novel KDM4 inhibitors.
The role of neuromonitoring in cardiovascular surgery Edmonds, Harvey L.; Rodriguez, Rosendo A.; Audenaert, Steve M. ...
Journal of cardiothoracic and vascular anesthesia,
1996, 1996-Jan, 1996-1-00, 19960101, Letnik:
10, Številka:
1
Journal Article
Recenzirano
This review describes the techniques currently used for quantitative neurophysiologic measurement during cardiac surgery and their potential impact on clinical outcome. Electroencephalography (EEG) ...characterizes cerebrocortical neuronal electrical activity and was part of some of the earliest cardiopulmonary bypass procedures, yet today it is not in widespread use. Each of the common misunderstandings regarding a supposed limitation of this technology is explained. Its major genuine shortcoming, a lack of selectivity, may now be overcome with the combined use of additional monitoring modalities. The influence of intracranial hemodynamics on observed EEG changes may be determined continuously and noninvasively with transcranial Doppler (TCD) ultrasound. TCD provides an indication of sudden change in either blood flow or vascular resistance as well as the detection of emboli. In addition, the metabolic status of cortical neurons can be monitored by regional cerebral venous oxygen saturation (rCVOS) using noninvasive transcranial near-infrared spectroscopy. The % rCVOS tends to remain remarkably stable over a wide range of temperatures, perfusion pressures, and anesthetic states. Marked change in either direction signifies a serious imbalance between oxygen delivery and consumption. Measurement of rCVOS does not require blood flow, pulsatile or otherwise, so that it offers the only means of monitoring during circulatory arrest. By characterizing the dynamic interplay among cerebral hemodynamics, metabolism, and electrogenesis, these technologies permit the rapid detection and correction of potentially hazardous conditions.
In chapter 1, we discuss the development of an ubiquitin-based proximity tagger for cell surface interactomics. Despite tremendous progress in developing technologies for unbiased analysis of ...protein-protein interactions for soluble proteins, technologies for cell surface ”interactomics” has proven more challenging. This is largely due to a lack of good proteomic tools for identifying weak, transient, and lipophilic complexes. However, due to the abundance of mammalian genes that encode for membrane proteins and the large number of drug targets that are found at the membrane, the ability to explore protein-protein and drug-protein interactions in this context has significant therapeutic importance. We describe the development of a tool we call the ”Ubiquitron”, which utilizes components of the endogenous ubiquitination system to label targets on the surface of intact cells with biotinylated ubiquitin, and does so in a minimally biased fashion with great sensitivity. We validate the speed and sensitivity of the tagger in a growth hormone/GHR system, and show that at least in a model system, targets can be unambiguously identified in a GFP-based model system by affinity purification-tandem mass spectrometry (AP-MS/MS). We also describe experiments where the tagger can be genetically fused to a receptor on the cell surface, and show that this ”Receptortron” can be used to assay the strength of interaction between two receptors. We envision using the ”Ubiquitron” to rapidly identify a partially- or fully-orphan ligand’s interactome, in contexts from phenotypic selections in phage display to identifying the targets of autoimmune antibodies. In chapter 2, we turn to the development of a tool called ”PhaNGS” or Phage-antibody Next Generation Sequencing, a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies. Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. Using 144 pre-selected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B-cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL), and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1, dominate the response to similar oncogenic perturbations in B-cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNAseq for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.
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