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1.
Nat Commun ; 12(1): 761, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536412

RESUMEN

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.


Asunto(s)
Arginina/metabolismo , Membrana Celular/metabolismo , Fusión de Membrana , Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Animales , Arginina/química , Sitios de Unión , Calcio/metabolismo , Neurotransmisores/metabolismo , Unión Proteica , Dominios Proteicos , Ratas , Proteínas SNARE/química , Sinaptotagmina I/química
2.
Nat Commun ; 12(1): 837, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547281

RESUMEN

Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.


Asunto(s)
/prevención & control , Evolución Molecular , Glicoproteína de la Espiga del Coronavirus/genética , /metabolismo , Animales , Unión Competitiva , /virología , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Pandemias , Unión Proteica , Dominios Proteicos , /fisiología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
3.
Nat Commun ; 12(1): 848, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558493

RESUMEN

The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.


Asunto(s)
/metabolismo , Mutación , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del Virus , Unión Competitiva , /virología , Humanos , Modelos Moleculares , Pandemias , Unión Proteica , Dominios Proteicos , Receptores Virales/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
4.
mSphere ; 6(1)2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33568452

RESUMEN

Compared to other human coronaviruses, the genetic diversity and evolution of human coronavirus 229E (HCoV-229E) are relatively understudied. We report a fatal case of COVID-19 pneumonia coinfected with HCoV-229E in Hong Kong. Genome sequencing of SARS-CoV-2 and HCoV-229E from a nasopharyngeal sample of the patient showed that the SARS-CoV-2 strain HK13 was most closely related to SARS-CoV-2 type strain Wuhan-Hu-1 (99.99% nucleotide identity), compatible with his recent history of travel to Wuhan. The HCoV-229E strain HK20-42 was most closely related to HCoV-229E strain SC0865 from the United States (99.86% nucleotide identity). To investigate if it may represent a newly emerged HCoV-229E genotype in Hong Kong, we retrieved 41 archived respiratory samples that tested positive for HCoV-229E from 2004 to 2019. Pneumonia and exacerbations of chronic airway diseases were common among infected patients. Complete RdRp, S, and N gene sequencing of the 41 HCoV-229E strains revealed that our contemporary HCoV-229E strains have undergone significant genetic drift with clustering of strains in chronological order. Two novel genogroups were identified, in addition to previously described genogroups 1 to 4, with recent circulating strains including strain HK20-42 belonging to novel genogroup 6. Positive selection was detected in the spike protein and receptor-binding domain, which may be important for viral evolution at the receptor-binding interphase. Molecular dating analysis showed that HCoV-229E shared the most recent common ancestor with bat and camel/alpaca 229E-related viruses at ∼1884, while camel/alpaca viruses had a relatively recent common ancestor at ∼1999. Further studies are required to ascertain the evolutionary origin and path of HCoV-229E.IMPORTANCE Since its first appearance in the 1960s, the genetic diversity and evolution of human coronavirus 229E (HCoV-229E) have been relatively understudied. In this study, we report a fatal case of COVID-19 coinfected with HCoV-229E in Hong Kong. Genome sequencing revealed that our SARS-CoV-2 strain is highly identical to the SARS-CoV-2 strain from Wuhan, compatible with the patient's recent travel history, whereas our HCoV-229E strain in this study is highly identical to a recent strain in the United States. We also retrieved 41 archived HCoV-229E strains from 2004 to 2019 in Hong Kong for sequence analysis. Pneumonia and exacerbations of chronic airway diseases were common diagnoses among the 41 patients. The results showed that HCoV-229E was evolving in chronological order. Two novel genogroups were identified in addition to the four preexisting HCoV-229E genogroups, with recent circulating strains belonging to novel genogroup 6. Molecular clock analysis dated bat-to-human and bat-to-camelid transmission to as early as 1884.


Asunto(s)
/patología , Resfriado Común/patología , Coronavirus Humano 229E/genética , Variación Genética/genética , /genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Niño , Preescolar , Coinfección/virología , Evolución Molecular , Femenino , Genoma Viral/genética , Hong Kong , Humanos , Lactante , Masculino , Persona de Mediana Edad , Dominios Proteicos/genética , Análisis de Secuencia de ARN , Glicoproteína de la Espiga del Coronavirus/genética , Adulto Joven
5.
Nat Commun ; 12(1): 793, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542240

RESUMEN

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted - and in some cases even shown - to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.


Asunto(s)
Adenosina Desaminasa/metabolismo , Elementos Alu/genética , Dominios Proteicos , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/aislamiento & purificación , Adenosina Desaminasa/ultraestructura , Dicroismo Circular , Inmunidad Innata , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Motivo de Reconocimiento de ARN , ARN Ribosómico/genética , ARN Ribosómico/inmunología , ARN Ribosómico/ultraestructura , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura
6.
Nat Commun ; 12(1): 887, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563969

RESUMEN

Polycomb repressive complex 1 (PRC1) is an essential chromatin-based repressor of gene transcription. How PRC1 engages with chromatin to identify its target genes and achieve gene repression remains poorly defined, representing a major hurdle to our understanding of Polycomb system function. Here, we use genome engineering and single particle tracking to dissect how PRC1 binds to chromatin in live mouse embryonic stem cells. We observe that PRC1 is highly dynamic, with only a small fraction stably interacting with chromatin. By integrating subunit-specific dynamics, chromatin binding, and abundance measurements, we discover that PRC1 exhibits low occupancy at target sites. Furthermore, we employ perturbation approaches to uncover how specific components of PRC1 define its kinetics and chromatin binding. Together, these discoveries provide a quantitative understanding of chromatin binding by PRC1 in live cells, suggesting that chromatin modification, as opposed to PRC1 complex occupancy, is central to gene repression.


Asunto(s)
Complejo Represivo Polycomb 1/química , Complejo Represivo Polycomb 1/metabolismo , Imagen Individual de Molécula , Animales , Sitios de Unión , Cromatina/química , Cromatina/metabolismo , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
7.
Nat Commun ; 12(1): 972, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563978

RESUMEN

Among the many questions unanswered for the COVID-19 pandemic are the origin of SARS-CoV-2 and the potential role of intermediate animal host(s) in the early animal-to-human transmission. The discovery of RaTG13 bat coronavirus in China suggested a high probability of a bat origin. Here we report molecular and serological evidence of SARS-CoV-2 related coronaviruses (SC2r-CoVs) actively circulating in bats in Southeast Asia. Whole genome sequences were obtained from five independent bats (Rhinolophus acuminatus) in a Thai cave yielding a single isolate (named RacCS203) which is most related to the RmYN02 isolate found in Rhinolophus malayanus in Yunnan, China. SARS-CoV-2 neutralizing antibodies were also detected in bats of the same colony and in a pangolin at a wildlife checkpoint in Southern Thailand. Antisera raised against the receptor binding domain (RBD) of RmYN02 was able to cross-neutralize SARS-CoV-2 despite the fact that the RBD of RacCS203 or RmYN02 failed to bind ACE2. Although the origin of the virus remains unresolved, our study extended the geographic distribution of genetically diverse SC2r-CoVs from Japan and China to Thailand over a 4800-km range. Cross-border surveillance is urgently needed to find the immediate progenitor virus of SARS-CoV-2.


Asunto(s)
Quirópteros/virología , /fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/sangre , Asia Sudoriental , Quirópteros/sangre , Geografía , Pruebas de Neutralización , Filogenia , Dominios Proteicos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo
8.
Commun Biol ; 4(1): 228, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589648

RESUMEN

SARS-CoV-2 has been mutating since it was first sequenced in early January 2020. Here, we analyze 45,494 complete SARS-CoV-2 geneome sequences in the world to understand their mutations. Among them, 12,754 sequences are from the United States. Our analysis suggests the presence of four substrains and eleven top mutations in the United States. These eleven top mutations belong to 3 disconnected groups. The first and second groups consisting of 5 and 8 concurrent mutations are prevailing, while the other group with three concurrent mutations gradually fades out. Moreover, we reveal that female immune systems are more active than those of males in responding to SARS-CoV-2 infections. One of the top mutations, 27964C > T-(S24L) on ORF8, has an unusually strong gender dependence. Based on the analysis of all mutations on the spike protein, we uncover that two of four SASR-CoV-2 substrains in the United States become potentially more infectious.


Asunto(s)
/virología , Mutación/genética , /genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , /metabolismo , Evolución Molecular , Femenino , Humanos , Masculino , Modelos Moleculares , Nucleocápside/metabolismo , Sistemas de Lectura Abierta/genética , Polimorfismo de Nucleótido Simple/genética , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Termodinámica , Estados Unidos
9.
Nat Commun ; 12(1): 1045, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594072

RESUMEN

Recurring chromosomal translocation t(10;17)(p15;q21) present in a subset of human acute myeloid leukemia (AML) patients creates an aberrant fusion gene termed ZMYND11-MBTD1 (ZM); however, its function remains undetermined. Here, we show that ZM confers primary murine hematopoietic stem/progenitor cells indefinite self-renewal capability ex vivo and causes AML in vivo. Genomics profilings reveal that ZM directly binds to and maintains high expression of pro-leukemic genes including Hoxa, Meis1, Myb, Myc and Sox4. Mechanistically, ZM recruits the NuA4/Tip60 histone acetyltransferase complex to cis-regulatory elements, sustaining an active chromatin state enriched in histone acetylation and devoid of repressive histone marks. Systematic mutagenesis of ZM demonstrates essential requirements of Tip60 interaction and an H3K36me3-binding PWWP (Pro-Trp-Trp-Pro) domain for oncogenesis. Inhibitor of histone acetylation-'reading' bromodomain proteins, which act downstream of ZM, is efficacious in treating ZM-induced AML. Collectively, this study demonstrates AML-causing effects of ZM, examines its gene-regulatory roles, and reports an attractive mechanism-guided therapeutic strategy.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Co-Represoras/química , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Leucemia Mieloide Aguda/patología , Lisina Acetiltransferasa 5/metabolismo , Acetilación , Animales , Carcinogénesis , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos/genética , Regulación Leucémica de la Expresión Génica , Genoma Humano , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Ratones Endogámicos BALB C , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Dominios Proteicos , Factores de Transcripción/metabolismo
10.
Genes (Basel) ; 12(2)2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572725

RESUMEN

SARS-CoV-2 is a recently emerged, novel human coronavirus responsible for the currently ongoing COVID-19 pandemic. Recombination is a well-known evolutionary strategy of coronaviruses, which may frequently result in significant genetic alterations, such as deletions throughout the genome. In this study we identified a co-infection with two genetically different SARS-CoV-2 viruses within a single patient sample via amplicon-based next generation sequencing in Hungary. The recessive strain contained an 84 base pair deletion in the receptor binding domain of the spike protein gene and was found to be gradually displaced by a dominant non-deleterious variant over-time. We have identified the region of the receptor-binding domain (RBD) that is affected by the mutation, created homology models of the RBDΔ84 mutant, and based on the available experimental data and calculations, we propose that the mutation has a deteriorating effect on the binding of RBD to the angiotensin-converting enzyme 2 (ACE2) receptor, which results in the negative selection of this variant. Extending the sequencing capacity toward the discovery of emerging recombinant or deleterious strains may facilitate the early recognition of novel strains with altered phenotypic attributes and understanding of key elements of spike protein evolution. Such studies may greatly contribute to future therapeutic research and general understanding of genomic processes of the virus.


Asunto(s)
/metabolismo , /metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , /virología , Línea Celular , Chlorocebus aethiops , Simulación por Computador , Humanos , Pandemias , Unión Proteica , Dominios Proteicos , Eliminación de Secuencia , Células Vero
11.
Molecules ; 26(3)2021 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-33525415

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection inducing coronavirus disease 2019 (COVID-19) is still an ongoing challenge. To date, more than 95.4 million have been infected and more than two million deaths have been officially reported by the WHO. Angiotensin-converting enzyme (ACE) plays a key role in the disease pathogenesis. In this computational study, seventeen coding variants were found to be important for ACE2 binding with the coronavirus spike protein. The frequencies of these allele variants range from 3.88 × 10-3 to 5.47 × 10-6 for rs4646116 (K26R) and rs1238146879 (P426A), respectively. Chloroquine (CQ) and its metabolite hydroxychloroquine (HCQ) are mainly used to prevent and treat malaria and rheumatic diseases. They are also used in several countries to treat SARS-CoV-2 infection inducing COVID-19. Both CQ and HCQ were found to interact differently with the various ACE2 domains reported to bind with coronavirus spike protein. A molecular docking approach revealed that intermolecular interactions of both CQ and HCQ exhibited mediation by ACE2 polymorphism. Further explorations of the relationship and the interactions between ACE2 polymorphism and CQ/HCQ would certainly help to better understand the COVID-19 management strategies, particularly their use in the absence of specific vaccines or drugs.


Asunto(s)
Cloroquina/química , Hidroxicloroquina/química , Simulación del Acoplamiento Molecular , Polimorfismo Genético , Glicoproteína de la Espiga del Coronavirus , /química , /metabolismo , /metabolismo , Cloroquina/farmacocinética , Cloroquina/uso terapéutico , Humanos , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/uso terapéutico , Dominios Proteicos , /metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
12.
PLoS One ; 16(2): e0242890, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33529230

RESUMEN

The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.


Asunto(s)
Expresión Génica , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Transfección/métodos , Animales , Células CHO , Cricetulus , Vectores Genéticos , Humanos , Factor 1 de Elongación Peptídica/genética , Plásmidos , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación
13.
Nat Commun ; 12(1): 867, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558520

RESUMEN

Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by lovastatin nonaketide synthase (LovB), with the assistance of a separate trans-acting enoyl reductase (LovC). A full DML synthesis comprises 8 polyketide synthetic cycles with about 35 steps. The assembling of the LovB-LovC complex, and the structural basis for the iterative and yet permutative functions of the megasynthase have remained a mystery. Here, we present the cryo-EM structures of the LovB-LovC complex at 3.60 Å and the core LovB at 2.91 Å resolution. The domain organization of LovB is an X-shaped face-to-face dimer containing eight connected domains. The binding of LovC laterally to the malonyl-acetyl transferase domain allows the completion of a L-shaped catalytic chamber consisting of six active domains. This architecture and the structural details of the megasynthase provide the basis for the processing of the intermediates by the individual catalytic domains. The detailed architectural model provides structural insights that may enable the re-engineering of the megasynthase for the generation of new statins.


Asunto(s)
Lovastatina/biosíntesis , Lovastatina/química , Biocatálisis , Modelos Moleculares , Naftalenos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/ultraestructura , Dominios Proteicos , Especificidad por Sustrato
14.
Nat Commun ; 12(1): 28, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397924

RESUMEN

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.


Asunto(s)
Núcleo Celular/metabolismo , Factores de Transcripción SOXB1/química , Factores de Transcripción SOXB1/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Drosophila/metabolismo , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Proteínas Mutantes/metabolismo , Células-Madre Neurales/metabolismo , Señales de Localización Nuclear/metabolismo , Mutación Puntual/genética , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/metabolismo , Factores de Transcripción SOXB1/genética , Relación Estructura-Actividad
15.
Nat Commun ; 12(1): 244, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431842

RESUMEN

The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.


Asunto(s)
Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , /química , /virología , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , /genética , Glicoproteína de la Espiga del Coronavirus/genética
16.
Nat Commun ; 12(1): 234, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431897

RESUMEN

Parasitoids are ubiquitous in natural ecosystems. Parasitic strategies are highly diverse among parasitoid species, yet their underlying genetic bases are poorly understood. Here, we focus on the divergent adaptation of a specialist and a generalist drosophilid parasitoids. We find that a novel protein (Lar) enables active immune suppression by lysing the host lymph glands, eventually leading to successful parasitism by the generalist. Meanwhile, another novel protein (Warm) contributes to a passive strategy by attaching the laid eggs to the gut and other organs of the host, leading to incomplete encapsulation and helping the specialist escape the host immune response. We find that these diverse parasitic strategies both originated from lateral gene transfer, followed with duplication and specialization, and that they might contribute to the shift in host ranges between parasitoids. Our results increase our understanding of how novel gene functions originate and how they contribute to host adaptation.


Asunto(s)
Proteínas de Insectos/metabolismo , Parásitos/fisiología , Estructuras Animales/metabolismo , Animales , Drosophila/parasitología , Genoma de los Insectos , Especificidad del Huésped , Interacciones Huésped-Parásitos , Inmunidad , Masculino , Mucinas/química , Filogenia , Dominios Proteicos , Especificidad de la Especie , Avispas/genética , Avispas/inmunología , Avispas/fisiología
17.
Nat Commun ; 12(1): 296, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436600

RESUMEN

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.


Asunto(s)
Lípidos/química , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Ingeniería de Proteínas , Acilación , Secuencia de Aminoácidos , Lipopéptidos/química , Lipopéptidos/metabolismo , Modelos Moleculares , Mutación Puntual/genética , Dominios Proteicos , Especificidad por Sustrato
18.
Nat Commun ; 12(1): 339, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436626

RESUMEN

Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.


Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa/química , Proteína 1 del Complejo de la Esclerosis Tuberosa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/química , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Biocatálisis , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteína 1 del Complejo de la Esclerosis Tuberosa/ultraestructura , Proteína 2 del Complejo de la Esclerosis Tuberosa/ultraestructura
19.
Nat Commun ; 12(1): 308, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436632

RESUMEN

Accumulating evidence shows that RAGE has an important function in the pathogenesis of sepsis. However, the mechanisms by which RAGE transduces signals to downstream kinase cascades during septic shock are not clear. Here, we identify SLP76 as a binding partner for the cytosolic tail of RAGE both in vitro and in vivo and demonstrate that SLP76 binds RAGE through its sterile α motif (SAM) to mediate downstream signaling. Genetic deficiency of RAGE or SLP76 reduces AGE-induced phosphorylation of p38 MAPK, ERK1/2 and IKKα/ß, as well as cytokine release. Delivery of the SAM domain into macrophages via the TAT cell-penetrating peptide blocks proinflammatory cytokine production. Furthermore, administration of TAT-SAM attenuates inflammatory cytokine release and tissue damage in mice subjected to cecal ligation and puncture (CLP) and protects these mice from the lethality of sepsis. These findings reveal an important function for SLP76 in RAGE-mediated pro-inflammatory signaling and shed light on the development of SLP76-targeted therapeutics for sepsis.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Terapia Molecular Dirigida , Fosfoproteínas/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Sepsis/tratamiento farmacológico , Animales , Bacteriófago T7/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Células HEK293 , Humanos , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Péptidos/metabolismo , Unión Proteica , Dominios Proteicos , Células RAW 264.7 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor para Productos Finales de Glicación Avanzada/química , Sepsis/patología , Transducción de Señal
20.
Science ; 371(6526): 284-288, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33446556

RESUMEN

The ability for viruses to mutate and evade the human immune system and cause infection, called viral escape, remains an obstacle to antiviral and vaccine development. Understanding the complex rules that govern escape could inform therapeutic design. We modeled viral escape with machine learning algorithms originally developed for human natural language. We identified escape mutations as those that preserve viral infectivity but cause a virus to look different to the immune system, akin to word changes that preserve a sentence's grammaticality but change its meaning. With this approach, language models of influenza hemagglutinin, HIV-1 envelope glycoprotein (HIV Env), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike viral proteins can accurately predict structural escape patterns using sequence data alone. Our study represents a promising conceptual bridge between natural language and viral evolution.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , VIH-1/genética , Virus de la Influenza A/genética , Gripe Humana/inmunología , /genética , Síndrome de Inmunodeficiencia Adquirida/virología , Sitios de Unión , Evolución Molecular , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Gripe Humana/virología , Mutación , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética
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