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As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an 'ETGE'-like degron-dependent manner. Cas9-'ETGE'-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-'ETGE'-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
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Current treatments for type 1 diabetes (T1D) focus on insulin replacement. We demonstrate the therapeutic potential of a secreted protein fraction from embryonic brown adipose tissue (BAT), independent of insulin. The large molecular weight secreted fraction mediates insulin receptor-dependent recovery of euglycemia in a T1D animal model, nonobese diabetic (NOD) mice, by suppressing glucagon secretion. This fraction also promotes white adipocyte differentiation and browning, maintains healthy BAT, and enhances glucose uptake in adipose tissue, skeletal muscle, and liver. From this fraction, we identify nidogen-2 as a critical BAT-secreted protein that reverses hyperglycemia in NOD mice, inhibits glucagon secretion from pancreatic α-cells, and mimics other actions of the entire secreted fraction. These findings confirm that BAT transplants affect physiology and demonstrate that BAT-secreted peptides represent a novel therapeutic approach to diabetes management. Furthermore, our research reveals a novel signaling role for nidogen-2, beyond its traditional classification as an extracellular matrix protein. HIGHLIGHTS: The large molecular weight brown adipocyte-secreted protein fraction suppresses glucagon secretion and normalizes glycemia in mouse models of type 1 diabetes (T1D), independent of insulin, offering a novel therapeutic strategy for disease management.Nidogen-2, a critical component of this fraction, is identified as an inhibitor of glucagon secretion in pancreatic α-cells by regulating intracellular messenger activities.The large-secreted protein fraction prevents T1D-related whitening of brown adipose tissue, promotes adipocyte differentiation, and enhances browning of inguinal white adipose tissue.This fraction enhances glucose uptake in adipose tissue, skeletal muscle, and liver through an insulin receptor-dependent pathway.
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Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of ~20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2D's central role in proteome management.
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Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.
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Enzima de Conversão de Angiotensina 2 , COVID-19 , Proteínas de Membrana , Nucleotidiltransferases , SARS-CoV-2 , Transdução de Sinais , Replicação Viral , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Humanos , SARS-CoV-2/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , COVID-19/imunologia , COVID-19/virologia , COVID-19/metabolismo , Linhagem Celular , Interferons/metabolismo , Imunidade Inata , Animais , Interferon Tipo I/metabolismoRESUMO
BACKGROUND: Plasmodium falciparum, the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-based combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs, such as lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasite's growing resistance to existing therapies. In this study, a common characteristic of the P. falciparum proteome-stretches of poly-lysine residues, such as those found in proteins related to adhesion and pathogenicity-is investigated for its potential to treat infected erythrocytes. METHODS: This study utilizes in vitro culturing of intra-erythrocytic P. falciparum to assess the ability of poly-lysine peptides to inhibit the parasite's growth, measured via flow cytometry of acridine orange-stained infected erythrocytes. The inhibitory effect of many poly-lysine lengths and modifications were tested this way. Affinity pull-downs and mass spectrometry were performed to identify the proteins interacting with these poly-lysines. RESULTS: A single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 h. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers retains or increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, affinity pull-downs and mass-spectrometry identify P. falciparum's outer membrane proteins as likely targets for polybasic peptide medications. CONCLUSION: Since poly-lysine dendrimers are already FDA-approved for drug delivery and this study displays their potency against intraerythrocytic P. falciparum, their adaptation as anti-malarial drugs presents a promising new therapeutic strategy for malaria.
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Antimaláricos , Eritrócitos , Plasmodium falciparum , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/farmacologia , Antimaláricos/química , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Peptídeos/farmacologia , Peptídeos/química , Humanos , Polímeros/farmacologia , Polímeros/química , Polilisina/farmacologia , Polilisina/químicaRESUMO
Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.
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Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. Interaction with CPI-motif peptides induced conformations within CP that bring the cap and stalk closer, while interaction with V-1 moves them away from one another. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wild-type (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.
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Proteínas de Capeamento de Actina , Actinas , Proteínas de Capeamento de Actina/química , Ligação Proteica , Regulação Alostérica , Actinas/metabolismo , Peptídeos/químicaRESUMO
Metastatic uveal melanoma (UM) patients typically survive only 2 to 3 years because effective therapy does not yet exist. Here, to facilitate the discovery of therapeutic targets in UM, we have identified protein kinase signaling mechanisms elicited by the drivers in 90% of UM tumors: mutant constitutively active G protein α-subunits encoded by GNAQ (Gq) or GNA11 (G11). We used the highly specific Gq/11 inhibitor FR900359 (FR) to elucidate signaling networks that drive proliferation, metabolic reprogramming, and dedifferentiation of UM cells. We determined the effects of FR on the proteome and phosphoproteome of UM cells as indicated by bioinformatic analyses with CausalPath and site-specific gene set enrichment analysis. We found that inhibition of oncogenic Gq/11 caused deactivation of PKC, Erk, and the cyclin-dependent kinases CDK1 and CDK2 that drive proliferation. Inhibition of oncogenic Gq/11 in UM cells with low metastatic risk relieved inhibitory phosphorylation of polycomb-repressive complex subunits that regulate melanocytic redifferentiation. Site-specific gene set enrichment analysis, unsupervised analysis, and functional studies indicated that mTORC1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 drive metabolic reprogramming in UM cells. Together, these results identified protein kinase signaling networks driven by oncogenic Gq/11 that regulate critical aspects of UM cell biology and provide targets for therapeutic investigation.
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Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Neoplasias Uveais , Humanos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/farmacologia , Proliferação de Células , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , Proteína Quinase C/metabolismo , Biologia Computacional , MutaçãoRESUMO
The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carcinoma de Células Escamosas/metabolismo , Fator 2 Relacionado a NF-E2 , Proteômica , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Biomarcadores Tumorais/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/uso terapêutico , FormaldeídoRESUMO
Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.
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The nucleosome acidic patch is a major interaction hub for chromatin, providing a platform for enzymes to dock and orient for nucleosome-targeted activities. To define the molecular basis of acidic patch recognition proteome wide, we performed an amino acid resolution acidic patch interactome screen. We discovered that the histone H3 lysine 36 (H3K36) demethylase KDM2A, but not its closely related paralog, KDM2B, requires the acidic patch for nucleosome binding. Despite fundamental roles in transcriptional repression in health and disease, the molecular mechanisms governing nucleosome substrate specificity of KDM2A/B, or any related JumonjiC (JmjC) domain lysine demethylase, remain unclear. We used a covalent conjugate between H3K36 and a demethylase inhibitor to solve cryogenic electron microscopy structures of KDM2A and KDM2B trapped in action on a nucleosome substrate. Our structures show that KDM2-nucleosome binding is paralog specific and facilitated by dynamic nucleosomal DNA unwrapping and histone charge shielding that mobilize the H3K36 sequence for demethylation.
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Lisina , Nucleossomos , Histonas/metabolismo , Cromatina , Histona Desmetilases com o Domínio Jumonji/químicaRESUMO
INTRODUCTION: Public-facing maps of COVID-19 cases, hospital admissions, and deaths are commonly displayed at the state, county, and zip code levels, and low case counts are suppressed to protect confidentiality. Public health authorities are tasked with case identification, contact tracing, and canvasing for educational purposes during a pandemic. Given limited resources, authorities would benefit from the ability to tailor their efforts to a particular neighborhood or congregate living facility. METHODS: We describe the methods of building a real-time visualization of patients with COVID-19-positive tests, which facilitates timely public health response to the pandemic. We developed an interactive street-level visualization that shows new cases developing over time and resolving after 14 days of infection. Our source data included patient demographics (ie, age, race and ethnicity, and sex), street address of residence, respiratory test results, and date of test. RESULTS: We used colored dots to represent infections. The resulting animation shows where new cases developed in the region and how patterns changed over the course of the pandemic. Users can enlarge specific areas of the map and see street-level detail on residential location of each case and can select from demographic overlays and contour mapping options to see high-level patterns and associations with demographics and chronic disease prevalence as they emerge. CONCLUSIONS: Before the development of this tool, local public health departments in our region did not have a means to map cases of disease to the street level and gain real-time insights into the underlying population where hotspots had developed. For privacy reasons, this tool is password-protected and not available to the public. We expect this tool to prove useful to public health departments as they navigate not only COVID-19 pandemic outcomes but also other public health threats, including chronic diseases and communicable disease outbreaks.
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COVID-19/epidemiologia , Pandemias , Saúde Pública/métodos , Doença Crônica/epidemiologia , Busca de Comunicante/métodos , Demografia/métodos , Surtos de Doenças/estatística & dados numéricos , Hospitalização , Humanos , Saúde Pública/estatística & dados numéricosRESUMO
Aberrant activation or suppression of WNT/ß-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the ß-catenin-dependent and ß-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated ß-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and ß-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.
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Caseína Quinase I , Via de Sinalização Wnt , beta Catenina , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fosforilação , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.
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Proteínas de Bactérias/antagonistas & inibidores , Carcinógenos/antagonistas & inibidores , Colite/prevenção & controle , Neoplasias Colorretais/prevenção & controle , Glucuronidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Triclosan/antagonistas & inibidores , Animais , Anti-Infecciosos Locais/química , Anti-Infecciosos Locais/metabolismo , Anti-Infecciosos Locais/toxicidade , Anticarcinógenos/química , Anticarcinógenos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biotransformação , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinógenos/química , Carcinógenos/metabolismo , Carcinógenos/toxicidade , Colite/induzido quimicamente , Colite/enzimologia , Colite/microbiologia , Colo/efeitos dos fármacos , Colo/microbiologia , Colo/patologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Expressão Gênica , Glucuronidase/química , Glucuronidase/genética , Glucuronidase/metabolismo , Inibidores de Glicosídeo Hidrolases/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triclosan/química , Triclosan/metabolismo , Triclosan/toxicidadeRESUMO
Homodimers are the most abundant type of enzyme in cells, and as such, they represent the most elemental system for studying the phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on a similar event at the second site, the most informative state is the asymmetric singly bound (lig1) form, yet it tends to be thermodynamically elusive. Here we obtain milligram quantities of lig1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of a highly purified, fully intact, active mixed labeled dimer, which provides the first glimpse of the lig1 intermediate. These data not only will make possible NMR-based investigations of allostery envisioned by us but also should facilitate other structural applications in which specific linkage of proteins is helpful.
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Cobre/química , Compostos Organometálicos/química , Alcinos/química , Sítio Alostérico , Azidas/química , Catálise , Reação de Cicloadição , Dimerização , Espectroscopia de Ressonância Magnética , Ligação Proteica , TermodinâmicaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.
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COVID-19/imunologia , COVID-19/metabolismo , Receptores Virais , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ciclo Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Perfilação da Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Proteômica , Receptores Virais/metabolismo , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Internalização do Vírus , Replicação ViralRESUMO
Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.
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Neuronal morphogenesis involves dramatic plasma membrane expansion, fueled by soluble N-ethylmaleimide-sensitive factor attachment protein eceptors (SNARE)-mediated exocytosis. Distinct fusion modes described at synapses include full-vesicle fusion (FVF) and kiss-and-run fusion (KNR). During FVF, lumenal cargo is secreted and vesicle membrane incorporates into the plasma membrane. During KNR, a transient fusion pore secretes cargo but closes without membrane addition. In contrast, fusion modes are not described in developing neurons. Here, we resolve individual exocytic events in developing murine cortical neurons and use classification tools to identify four distinguishable fusion modes: two FVF-like modes that insert membrane material and two KNR-like modes that do not. Discrete fluorescence profiles suggest distinct behavior of the fusion pore. Simulations and experiments agree that FVF-like exocytosis provides sufficient membrane material for morphogenesis. We find the E3 ubiquitin ligase TRIM67 promotes FVF-like exocytosis in part by limiting incorporation of the Qb/Qc SNARE SNAP47 into SNARE complexes and, thus, SNAP47 involvement in exocytosis.
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Proteínas do Citoesqueleto/metabolismo , Exocitose , Neurogênese , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Sinapses/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Feminino , Camundongos , Camundongos Knockout , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Sinapses/genética , Proteínas com Motivo Tripartido/genéticaRESUMO
Kinases form the backbone of numerous cell signaling pathways, with their dysfunction similarly implicated in multiple pathologies. Further facilitated by their druggability, kinases are a major focus of therapeutic development efforts in diseases such as cancer, infectious disease and autoimmune disorders. While their importance is clear, the role or biological function of nearly one-third of kinases is largely unknown. Here, we describe a data resource, the Dark Kinase Knowledgebase (DKK; https://darkkinome.org), that is specifically focused on providing data and reagents for these understudied kinases to the broader research community. Supported through NIH's Illuminating the Druggable Genome (IDG) Program, the DKK is focused on data and knowledge generation for 162 poorly studied or 'dark' kinases. Types of data provided through the DKK include parallel reaction monitoring (PRM) peptides for quantitative proteomics, protein interactions, NanoBRET reagents, and kinase-specific compounds. Higher-level data is similarly being generated and consolidated such as tissue gene expression profiles and, longer-term, functional relationships derived through perturbation studies. Associated web tools that help investigators interrogate both internal and external data are also provided through the site. As an evolving resource, the DKK seeks to continually support and enhance knowledge on these potentially high-impact druggable targets.