ABSTRACT
Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.
Subject(s)
COVID-19/metabolism , COVID-19/virology , Host-Pathogen Interactions , Lectins, C-Type/metabolism , Membrane Proteins/metabolism , Myeloid Cells/immunology , Myeloid Cells/metabolism , Neoplasm Proteins/metabolism , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/genetics , Cell Line , Cytokines , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Inflammation Mediators/metabolism , Lectins, C-Type/chemistry , Membrane Proteins/chemistry , Models, Molecular , Neoplasm Proteins/chemistry , Protein Binding , Protein Conformation , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity RelationshipABSTRACT
Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/ß-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease.
Subject(s)
Cell Adhesion Molecules/metabolism , Endoplasmic Reticulum/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Cell Adhesion Molecules/chemistry , Endoplasmic Reticulum/genetics , High-Throughput Nucleotide Sequencing , Humans , Intrinsically Disordered Proteins , Membrane Proteins/genetics , Oncogene Proteins/chemistry , Oncogene Proteins/metabolism , RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , Sequence Analysis, RNAABSTRACT
In order to directly observe the refolding kinetics from a partially misfolded state to a native state in the bottom of the protein-folding funnel, we used a "caging" strategy to trap the ß-sheet structure of ubiquitin in a misfolded conformation. We used molecular dynamics simulation to generate the cage-induced, misfolded structure and compared the structure of the misfolded ubiquitin with native ubiquitin. Using laser flash irradiation, the cage can be cleaved from the misfolded structure within one nanosecond, and we monitored the refolding kinetics of ubiquitin from this misfolded state to the native state by photoacoustic calorimetry and photothermal beam deflection techniques on nanosecond to millisecond timescales. Our results showed two refolding events in this refolding process. The fast event is shorter than 20 ns and corresponds to the instant collapse of ubiquitin upon cage release initiated by laser irradiation. The slow event is ~60 µs, derived from a structural rearrangement in ß-sheet refolding. The event lasts 10 times longer than the timescale of ß-hairpin formation for short peptides as monitored by temperature jump, suggesting that rearrangement of a ß-sheet structure from a misfolded state to its native state requires more time than ab initio folding of a ß-sheet.
Subject(s)
Ubiquitin/chemistry , Calorimetry , Humans , Kinetics , Least-Squares Analysis , Molecular Dynamics Simulation , Mutant Proteins/metabolism , Photoacoustic Techniques , Photolysis , Protein Folding , Structural Homology, Protein , Thermodynamics , Time FactorsABSTRACT
Signal sequence-encoding mRNAs undergo translation-dependent localization to the endoplasmic reticulum (ER) and at the ER are anchored via translation on Sec61-bound ribosomes. Recent investigations into the composition and membrane association characteristics of ER-associated mRNAs have, however, revealed both ribosome-dependent (indirect) and ribosome-independent (direct) modes of mRNA association with the ER. These findings raise important questions regarding our understanding of how mRNAs are selected, localized, and anchored to the ER. Using semi-intact tissue culture cells, we performed a polysome solubilization screen and identified conditions that distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. To gain insight into the molecular basis of direct mRNA anchoring to the ER, we performed RNA-protein UV photocross-linking studies in rough microsomes and demonstrate that numerous ER integral membrane proteins display RNA binding activity. Quantitative proteomic analyses of HeLa cytosolic and ER-bound polysome fractions identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins, whereas translocon accessory proteins, such as ribophorin I, were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocross-linked ER protein-RNA adducts identified Sec61α,ß and ribophorin I as ER-poly(A) mRNA-binding proteins, suggesting unexpected roles for the protein translocation and modification machinery in mRNA anchoring to the ER. In summary, we propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable an mRNA transcriptome-wide function for the ER in protein synthesis.
Subject(s)
Endoplasmic Reticulum/metabolism , Protein Transport/genetics , RNA, Messenger/genetics , Transcriptome/genetics , Endoplasmic Reticulum/genetics , HeLa Cells , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Polyribosomes/genetics , Protein Sorting Signals/genetics , RNA-Binding Proteins/biosynthesis , Ribosomes/genetics , SEC Translocation ChannelsABSTRACT
In this work, we found that, during storage or after UV irradiation, ThT is demethylated or oxidized, forming three derivatives. These three derivatives were purified by high performance liquid chromatography and characterized by mass and nuclear magnetic resonance spectroscopy and the spectroscopic properties of pure ThT and the derivatives carefully compared. Our results show that the emission peak at 450 nm results from oxidized ThT and not from the monomeric form of ThT, as previously proposed. The partial conversion of ThT into oxidized and demethylated derivatives has an effect on amyloid detection using ThT assay. Irradiated ThT has the same lag time as pure ThT in the amyloidogenesis of insulin, but the intensity of the emitted fluorescence is significantly decreased.
Subject(s)
Amyloid/chemistry , Thiazoles/chemistry , Ultraviolet Rays , Benzothiazoles , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Photochemical ProcessesABSTRACT
Kinetic measurement of protein folding is limited by the method used to trigger folding. Traditional methods, such as stopped flow, have a long mixing dead time and cannot be used to monitor fast folding processes. Here, we report a compound, 4-(bromomethyl)-6,7-dimethoxycoumarin, that can be used as a "photolabile cage" to study the early stages of protein folding. The folding process of a protein, RD1, including kinetics, enthalpy, and volume change, was studied by the combined use of a phototriggered caging strategy and time-resolved photoacoustic calorimetry. The cage caused unfolding of the photolabile protein, and then a pulse UV laser (â¼10(-9) s) was used to break the cage, leaving the protein free to refold and allowing the resolving of two folding events on a nanosecond time scale. This strategy is especially good for monitoring fast folding proteins that cannot be studied by traditional methods.