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Doud1,2,3,4 , Orr Ashenberg1,4 , and Jesse D. ... However, experiments have also shown that despite such epistasis, the amino-acid pref- erences of many sites are similar across homologs (Risso et al., 2015; Ashenberg et al., 2013; Serrano et ...doi:10.1101/018457 fatcat:cwycx5fw3bg3vexdlbzjbpupsq
Ashenberg. M.T.L. is an Early Career Scientist of the Howard Hughes Medical Institute. We used computer resources provided by National Science Foundation award 0821391. ...doi:10.1016/j.jmb.2011.08.011 pmid:21854787 pmcid:PMC3210482 fatcat:pu27hlzbjnfwzh4xsfsg22gxcu
The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza is more resistant than avian influenza to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonoticdoi:10.1101/071969 fatcat:o2hu72xp2vbgna4ou2gzszukdu
more »... sions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all ≈10,000 amino-acid mutations to NP affect MxA sensitivity. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect interactions with host factors.
Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-componentdoi:10.1016/j.cell.2008.04.040 pmid:18555780 pmcid:PMC2453690 fatcat:frhgiglfg5abln2auiza3m6elq
more »... naling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.
Ashenberg. M.T.L. is an Early Career Scientist of the Howard Hughes Medical Institute. We used computer resources provided by National Science Foundation award 0821391. ...doi:10.1016/j.jmb.2013.01.011 pmid:23333741 pmcid:PMC3636764 fatcat:zqjna7jisvfyxlhstba2czsf3i
The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza is more resistant than avian influenza to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonoticdoi:10.1371/journal.ppat.1006288 pmid:28346537 pmcid:PMC5383324 fatcat:bytlcwyamrehnnimaajkrvzvbi
more »... sions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all ≈10,000 amino-acid mutations to NP affect MxA sensitivity. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect interactions with host factors. Author Summary During viral infection, human cells express proteins that can restrict virus replication. However, in many cases it remains unclear what determines the sensitivity of a given viral strain to a particular restriction factor. Here we use a high-throughput approach to measure how all amino-acid mutations to the nucleoprotein of influenza virus affect restriction by the human protein MxA. We find several dozen sites where mutations substantially affect influenza's sensitivity to MxA. While a few of these sites are known to have fixed mutations during past adaptations of influenza to humans, most of the sites are broadly conserved across all influenza strains and have never previously been described as affecting MxA resistance. Our results therefore show that the known historical evolution of influenza has only involved substitutions at a small fraction of the sites where mutations can in principle affect MxA resistance. We suggest that this is because many sites are already broadly fixed at amino acids that confer high resistance.
However, experiments have also shown that despite such epistasis, the amino acid preferences of many sites are similar across homologs (Serrano et al. 1993; Ashenberg et al. 2013; Risso et al. 2015) . ... In addition, selection for protein stability is a major constraint on evolution (Bloom et al. 2005; DePristo et al. 2005) , and experiments on NP (Ashenberg et al. 2013 ) and other proteins (Serrano ...doi:10.1093/molbev/msv167 pmid:26226986 pmcid:PMC4626756 fatcat:ogspdunjl5eu3fvse6rpyiucyq
23 Running Title: Specificity of hybrid histidine kinases 24 25 2 Summary 26 Signal transduction proteins are often multidomain proteins that arose through the fusion of 27 previously independent proteins. How such a change in the spatial arrangement of proteins 28 impacts their evolution and the selective pressures acting on individual residues is largely 29 unknown. We explored this problem in the context of bacterial two-component signaling 30 pathways, which typically involve a sensordoi:10.1111/mmi.12064 pmid:23078131 fatcat:jbvkph3uxbgrppkvmsddshmklu
more »... ine kinase that specifically phosphorylates a 31 single cognate response regulator. Although usually found as separate proteins, these proteins 32 are sometimes fused into a so-called hybrid histidine kinase. Here, we demonstrate that the 33 isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer 34 specificity in vitro relative to canonical histidine kinases. However, hybrid kinases 35 phosphotransfer almost exclusively to their covalently attached response regulator domain, 36 whose effective concentration exceeds that of all soluble response regulators. These findings 37 indicate that the fused response regulator in a hybrid kinase normally prevents detrimental 38 cross-talk between pathways. More generally, our results shed light on how the spatial 39 properties of signaling pathways can significantly affect their evolution, with additional 40 implications for the design of synthetic signaling systems. 41 42
Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. Todoi:10.1371/journal.pgen.1001220 pmid:21124821 pmcid:PMC2991266 fatcat:kodowcjkwre4zivskeifakjwcq
more »... putative specificity-determining residues, we have analyzed amino acid coevolution in twocomponent proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highlyrelated response regulators and vice versa. Our results also suggest that the mutational trajectories taken by twocomponent signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.
T-cells are critical effector cells of cancer immunotherapies, but little is known about T-cell gene expression programs in diffuse gliomas. We leveraged single-cell RNA-seq to chart the gene expression and clonal landscape of tumor-infiltrating T-cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. Our analysis revealed subsets of T-cells that expressed several NK-cell receptors, in particular the inhibitory CD161 receptor (KLRB1 gene).doi:10.1093/noajnl/vdab070.059 fatcat:kjyokzmtkze7jcwqg3y2kbaray
more »... 1 was overexpressed by clonally expanded CD8 T-cells, and larger populations of T-cells expressed CD161 than PD-1. The CLEC2D ligand of CD161 was expressed by malignant cells and myeloid cells, and inactivation of KLRB1 enhanced anti-tumor T-cell function. KLRB1 was also expressed by substantial T-cell populations in multiple other human cancers. CD161 and other NK-cell receptors expressed by T-cells represent opportunities for immunotherapy of diffuse gliomas and other human cancers.
Massively parallel single-cell and single-nucleus RNA-seq (sc/snRNA-seq) have opened the way to systematic tissue atlases in health and disease, but as the scale of data generation is growing, so does the need for computational pipelines for scaled analysis. Here, we developed Cumulus, a cloud-based framework for analyzing large scale sc/snRNA-seq datasets. Cumulus combines the power of cloud computing with improvements in algorithm implementations to achieve high scalability, low cost,doi:10.1101/823682 fatcat:xw5om6lt4vhg7mc7thkh24xjk4
more »... endliness, and integrated support for a comprehensive set of features. We benchmark Cumulus on the Human Cell Atlas Census of Immune Cells dataset of bone marrow cells and show that it substantially improves efficiency over conventional frameworks, while maintaining or improving the quality of results, enabling large-scale studies.
A number of autoimmunity-associated MHC class II proteins interact only weakly with the invariant chain–derived class II–associated invariant chain peptide (CLIP). CLIP dissociates rapidly from I-Ag7 even in the absence of DM, and this property is related to the type 1 diabetes–associated β57 polymorphism. We generated knock-in non-obese diabetic (NOD) mice with a single amino acid change in the CLIP segment of the invariant chain in order to moderately slow CLIP dissociation from I-Ag7. Thesedoi:10.1084/jem.20180300 pmid:30185635 pmcid:PMC6170167 fatcat:ta5bwkbbmbdkziwydi3l6bq53q
more »... nock-in mice had a significantly reduced incidence of spontaneous type 1 diabetes and diminished islet infiltration by CD4 T cells, in particular T cells specific for fusion peptides generated by covalent linkage of proteolytic fragments within β cell secretory granules. Rapid CLIP dissociation enhanced the presentation of such extracellular peptides, thus bypassing the conventional MHC class II antigen-processing pathway. Autoimmunity-associated MHC class II polymorphisms therefore not only modify binding of self-peptides, but also alter the biochemistry of peptide acquisition.
The threat of viral pandemics demands a comprehensive understanding of evolution at the host-pathogen interface. Here, we show that the accessibility of adaptive mutations in influenza nucleoprotein at fever-like temperatures is mediated by host chaperones. Particularly noteworthy, we observe that the Pro283 nucleoprotein variant, which (doi:10.1371/journal.pbio.3000008 pmid:30222731 pmcid:PMC6160216 fatcat:dh4typqaxvgsfgzskgmcbrgm7y
Intrauterine growth restriction (IUGR) of fetuses affects 5-10% of pregnancies and is associated with perinatal morbidity, mortality and long-term health issues. Understanding genetic predisposition to IUGR is challenging, owing to extensive gene polymorphisms, linkage disequilibrium, and maternal and paternal influence. Here, we demonstrate that the inhibitory receptor, KIR2DL1, expressed on maternal uterine natural killer (uNK) cells, in interaction with the paternally-inherited HLA-C*05, andoi:10.1101/2021.03.26.437292 fatcat:ylrelyeiprfptm6g57cww7qpde
more »... LA-C group 2 allotype, expressed on fetal trophoblast cells, causes IUGR in a humanised mouse model. Micro-CT imaging of the uteroplacental vasculature revealed reduced uterine spiral artery diameter and increased segment length, increasing fetal blood flow resistance. Single cell RNA-Seq from the maternal-fetal interface highlighted expression programs activated by KIR2DL1-induced IUGR in several placental cell types, including degradation of extracellular matrix components, angiogenesis, and uNK cell communication, suggesting a complex response underlying IUGR. As current IUGR treatments are insufficient, our findings provide important insight for drug development.
During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone marrow derived hematopoietic progenitors that migrate through the bloodstream. In human, the nature of these thymus immigrants has remained unclear. Here, we employ single-cell RNA sequencing on approximately 70.000 CD34+ thymocytes to unravel the heterogeneity of the human immature postnatal thymocytes. Integration of bone marrow and peripheral blood precursors datasets identifies several putativedoi:10.1101/2020.04.07.007237 fatcat:2zncj3dgpbggvfehb2xyqau3nm
more »... hymus seeding precursors that display heterogeneity for currently used surface markers as revealed by CITEseq. Besides T cell precursors, we discover branches of intrathymic developing dendritic cells with predominantly plasmacytoid DCs. Trough trajectory inference, we delineate the transcriptional dynamics underlying early human T-lineage development from which we predict transcription factor modules that drive stage-specific steps of human T cell development. Thus, our work resolves the heterogeneity of thymus seeding precursors in human and reveals the molecular mechanisms that drive their in vivo cell fate.
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