RESUMEN
Despite unequivocal roles in disease, transcription factors (TFs) remain largely untapped as pharmacologic targets due to the challenges in targeting protein-protein and protein-DNA interactions. Here we report a chemical strategy to generate modular synthetic transcriptional repressors (STRs) derived from the bHLH domain of MAX. Our synthetic approach yields chemically stabilized tertiary domain mimetics that cooperatively bind the MYC/MAX consensus E-box motif with nanomolar affinity, exhibit specificity that is equivalent to or beyond that of full-length TFs and directly compete with MYC/MAX protein for DNA binding. A lead STR directly inhibits MYC binding in cells, downregulates MYC-dependent expression programs at the proteome level and inhibits MYC-dependent cell proliferation. Co-crystallization and structure determination of a STR:E-box DNA complex confirms retention of DNA recognition in a near identical manner as full-length bHLH TFs. We additionally demonstrate structure-blind design of STRs derived from alternative bHLH-TFs, confirming that STRs can be used to develop highly specific mimetics of TFs targeting other gene regulatory elements.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc , Factores de Transcripción , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Secuencias Hélice-Asa-Hélice , Secuencias Reguladoras de Ácidos Nucleicos , ADN/genética , ADN/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed to be essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found that human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system. IMPORTANCE SARS-CoV-2 is the third lethal respiratory coronavirus, after MERS-CoV and SARS-CoV, to emerge this century, causing millions of deaths worldwide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1α (IRE1α) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1α kinase and RNase activities, SARS-CoV-2 only partially activates IRE1α, promoting its kinase activity but not RNase activity. Based on IRE1α-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1α RNase activation as a strategy to limit detection by the host immune system.
Asunto(s)
COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Animales , Ratones , Humanos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Estrés del Retículo Endoplásmico/genética , SARS-CoV-2/genética , Inositol , Proteínas Serina-Treonina Quinasas/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Ribonucleasas/genética , Factores de Transcripción , ARN Mensajero , Pulmón/metabolismo , Interferones , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available, or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system. IMPORTANCE: SARS-CoV-2 is the third lethal respiratory coronavirus after MERS-CoV and SARS-CoV to emerge this century, causing millions of deaths world-wide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1α (IRE1α) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1α kinase and RNase activities, SARS-CoV-2 only partially activates IRE1α, promoting its kinase activity but not RNase activity. Based on IRE1α-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1α RNase activation as a strategy to limit detection by the host immune system.
RESUMEN
The remotely sensed burned area (BA) products can provide continuous and spatiotemporally explicit characteristics of fire patches, which are critical data sources for understanding regional fire regimes. However, their accuracy remains to be improved. In this study, a global BA product (i.e., CCI_Fire) at 250 m resolution was integrated with global forest change (GFC) product at 30 m to generate a refined BA product, named CCI_GFC product, whose accuracy was evaluated through comparing the BA with pre-existing fire patches data. To reveal the characteristics of forest fire in China between 2001 and 2017, we conducted a grid analysis at 0.05°×0.05° spatial resolution based on the refined BA product and the spatial pattern of eco-regions at the macro scale. The results showed that the accuracy metrics including the recognition rate (RR), variance explained (R2), root mean squared error (RMSE), mean absolute percentage error (MAPE) of the CCI_GFC product (i.e., 83%, 0.91, 0.28, and 8.5% respectively) were all superior to the CCI_Fire product (i.e., 74%, 0.86, 0.36, and 11.8% respectively) and the MCD64A1 product (i.e., 35%, 0.78, 0.48, and 17.3% respectively). The total burned area of forest was approximately 12.11 million hm2 for the whole country from 2001 to 2017, while the annual burned area temporally decreased. Forest fires in China were dominated by the low-frequency [0Asunto(s)
Incendios
, Incendios Forestales
, China
, Bosques
, Estaciones del Año
, Árboles
RESUMEN
Herein, we present a serendipitously discovered chemoselective labelling of protein N-homocysteinylation with bioorthogonal azide probes. The reaction proceeds rapidly under alkaline and heating conditions. Our experiments suggest that azides can be converted to aldehydes in situ catalyzed by heme(ii), followed by a condensation with protein N-homocysteinylation to afford stable 1,3-thiazines.
Asunto(s)
Azidas/química , Hemo/química , Homocisteína/análogos & derivados , Proteínas/química , Animales , Biotina/química , Catálisis , Bovinos , Homocisteína/química , Concentración de Iones de Hidrógeno , Mioglobina/química , Mioglobina/metabolismo , Péptidos/análisis , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
Hyperhomocysteinemia (HHcy) refers to a medical condition of abnormally high level of homocysteine (Hcy) in blood (>15 µmol L-1) and has been clinically implicated with cardiovascular diseases and neurodegenerative disorders. Excessive Hcy can be converted to a reactive thioester intermediate, Hcy thiolactone (HTL), which selectively reacts with protein lysine residues ("N-homocysteinylation") and this non-enzymatic modification largely contributes to manifestations of HHcy. However, the proteome-wide detection of protein N-homocysteinylation remains a challenge to date. In this work, we report a chemoselective reaction to label and enrich N-homocysteinylation from complex proteome samples as inspired by native chemical ligation for protein synthesis. Alkynyl thioester probes are synthesized and the reaction is validated with small molecule and purified protein models successfully. We performed quantitative chemical proteomics to identify more than 800 N-homocysteinylated proteins as well as 304 N-homocysteinylated sites directly from HTL-treated HeLa cells. The chemical proteomics strategies will facilitate functional study of protein N-homocysteinylations in the HHcy-implicated diseases.