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1.
Chemosphere ; 262: 128313, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33182081

RESUMEN

Androgens and androgen receptor regulate a variety of biological effects in the human body. The impaired functioning of androgen receptor may have different adverse health effects from cancer to infertility. Therefore, it is important to determine whether new chemicals have any binding activity and act as androgen agonists or antagonists before commercial use. Due to the large number of chemicals that require experimental testing, the computational methods are a viable alternative. Therefore, the aim of the present study was to develop predictive QSAR models for classifying compounds according to their activity at the androgen receptor. A large data set of chemicals from the CoMPARA project was used for this purpose and random forest classification models have been developed for androgen binding, agonistic, and antagonistic activity. In addition, a unique effort has been made for multi-class approach that discriminates between inactive compounds, agonists and antagonists simultaneously. For the evaluation set, the classification models predicted agonists with 80% of accuracy and for the antagonists' and binders' the respective metrics were 72% and 78%. Combining agonists, antagonists and inactive compounds into a multi-class approach added complexity to the modelling task and resulted to 64% prediction accuracy for the evaluation set. Considering the size of the training data sets and their imbalance, the achieved evaluation accuracy is very good. The final classification models are available for exploring and predicting at QsarDB repository (https://doi.org/10.15152/QDB.236).


Asunto(s)
Antagonistas de Receptores Androgénicos/clasificación , Andrógenos/clasificación , Modelos Químicos , Receptores Androgénicos/metabolismo , Antagonistas de Receptores Androgénicos/química , Antagonistas de Receptores Androgénicos/farmacología , Andrógenos/química , Andrógenos/farmacología , Humanos , Aprendizaje Automático , Unión Proteica , Relación Estructura-Actividad Cuantitativa
2.
Int J Mol Sci ; 21(21)2020 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-33158276

RESUMEN

Binding to the host receptor is a critical initial step for the coronavirus SARS-CoV-2 spike protein to enter into target cells and trigger virus transmission. A detailed dynamic and energetic view of the binding mechanisms underlying virus entry is not fully understood and the consensus around the molecular origins behind binding preferences of SARS-CoV-2 for binding with the angiotensin-converting enzyme 2 (ACE2) host receptor is yet to be established. In this work, we performed a comprehensive computational investigation in which sequence analysis and modeling of coevolutionary networks are combined with atomistic molecular simulations and comparative binding free energy analysis of the SARS-CoV and SARS-CoV-2 spike protein receptor binding domains with the ACE2 host receptor. Different from other computational studies, we systematically examine the molecular and energetic determinants of the binding mechanisms between SARS-CoV-2 and ACE2 proteins through the lens of coevolution, conformational dynamics, and allosteric interactions that conspire to drive binding interactions and signal transmission. Conformational dynamics analysis revealed the important differences in mobility of the binding interfaces for the SARS-CoV-2 spike protein that are not confined to several binding hotspots, but instead are broadly distributed across many interface residues. Through coevolutionary network analysis and dynamics-based alanine scanning, we established linkages between the binding energy hotspots and potential regulators and carriers of signal communication in the virus-host receptor complexes. The results of this study detailed a binding mechanism in which the energetics of the SARS-CoV-2 association with ACE2 may be determined by cumulative changes of a number of residues distributed across the entire binding interface. The central findings of this study are consistent with structural and biochemical data and highlight drug discovery challenges of inhibiting large and adaptive protein-protein interfaces responsible for virus entry and infection transmission.


Asunto(s)
Betacoronavirus/metabolismo , Infecciones por Coronavirus/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Infecciones por Coronavirus/enzimología , Infecciones por Coronavirus/virología , Interacciones Microbiota-Huesped , Humanos , Pandemias , Neumonía Viral/enzimología , Neumonía Viral/virología , Unión Proteica , Dominios Proteicos , Receptores Virales/metabolismo , Transducción de Señal , Internalización del Virus
3.
PLoS One ; 15(11): e0241168, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33170884

RESUMEN

The SARS-CoV-2 virion responsible for the current world-wide pandemic COVID-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD) within its distal, prefusion S1 domain. The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions (charge, partial charge, and van der Waals) of the SARS-CoV-2 Spike protein in its static prefusion state based on two recent and independent experimental structure publications. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences (different so-called "glue" points) between residues in the Up and Down state protomers. The S2 proximal, fusion domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer's intrachain, S1-S2 interactions. However, the S1 domain interactions across neighboring protomers, which include the RBD-NTD cross chain interactions, showed significant energetic differences between Up-Down and Down-Down neighboring protomers. This included, for example, a key RBD residue ARG357 in the Up-Down interaction and a three residue sequence ALA520-PRO521-ALA522, associated with a turn structure in the RBD of the Up state protomer, acting as a stabilizing interaction with the NTD of its neighbor protomer. Additionally, our intra chain dominant energy mappings within each protomer, identified a significant "glue" point or possible "latch" for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the amino acid R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-CoV-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. We conducted preliminary molecular dynamics simulations across 0.1 µ seconds to see if this latch provided structural stability and indeed found that a single point mutation (Q564G) resulted in the latch releasing transforming the protomer from the Down to the Up state conformation. Full trimeric Spike protein studies of the same mutation across all protomers, however, did not exhibit latch release demonstrating the critical importance of interchain interactions across the S1 domain, including RBD-NTD neighboring chain interactions. Therapies aimed at disrupting these noncovalent interactions could be a viable route for the physico-chemical mitigation of this deadly virion.


Asunto(s)
Betacoronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Humanos , Simulación de Dinámica Molecular , Pandemias , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , Mutación Puntual , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Termodinámica
4.
Nat Commun ; 11(1): 5559, 2020 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-33144569

RESUMEN

Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.


Asunto(s)
Colesterol/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Multimerización de Proteína , Proteínas de Unión al GTP rab/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas/genética , LDL-Colesterol/metabolismo , Endosomas/metabolismo , Endosomas/ultraestructura , Colorantes Fluorescentes/metabolismo , Genoma Humano , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Hidroximetilglutaril-CoA Sintasa/metabolismo , Lisosomas/ultraestructura , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Unión Proteica
5.
Elife ; 92020 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-33164751

RESUMEN

Pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus 19 disease (COVID-19) which presents a large spectrum of manifestations with fatal outcomes in vulnerable people over 70-years-old and with hypertension, diabetes, obesity, cardiovascular disease, COPD, and smoking status. Knowledge of the entry receptor is key to understand SARS-CoV-2 tropism, transmission and pathogenesis. Early evidence pointed to angiotensin-converting enzyme 2 (ACE2) as SARS-CoV-2 entry receptor. Here, we provide a critical summary of the current knowledge highlighting the limitations and remaining gaps that need to be addressed to fully characterize ACE2 function in SARS-CoV-2 infection and associated pathogenesis. We also discuss ACE2 expression and potential role in the context of comorbidities associated with poor COVID-19 outcomes. Finally, we discuss the potential co-receptors/attachment factors such as neuropilins, heparan sulfate and sialic acids and the putative alternative receptors, such as CD147 and GRP78.


Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Peptidil-Dipeptidasa A/fisiología , Neumonía Viral/virología , Acoplamiento Viral , Basigina/fisiología , Comorbilidad , Infecciones por Coronavirus/epidemiología , Regulación Enzimológica de la Expresión Génica , Heparitina Sulfato/fisiología , Humanos , Hipertensión/epidemiología , Hipertensión/fisiopatología , Neuropilina-1/fisiología , Oligopéptidos/fisiología , Especificidad de Órganos , Pandemias , Neumonía Viral/epidemiología , Unión Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Virales , Sistema Renina-Angiotensina/fisiología , Sistema Respiratorio/enzimología , Ácidos Siálicos/fisiología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/fisiología , Internalización del Virus
6.
Nat Commun ; 11(1): 5777, 2020 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-33188170

RESUMEN

Vibrio parahaemolyticus is the leading cause of seafood-borne diarrheal diseases. Experimental overproduction of a type 3 secretion system (T3SS1) in this pathogen leads to decreased intestinal colonization, which suggests that T3SS1 repression is required for maximal virulence. However, the mechanisms by which T3SS1 is repressed in vivo are unclear. Here, we show that host-derived nitrite modifies the activity of a bacterial histidine kinase and mediates T3SS1 repression. More specifically, nitrite activates histidine kinase sensor VbrK through S-nitrosylation on cysteine 86, which results in downregulation of the entire T3SS1 operon through repression of its positive regulator exsC. Replacement of cysteine 86 with a serine (VbrK C86S mutant) leads to increased expression of inflammatory cytokines in infected Caco-2 cells. In an infant rabbit model of infection, the VbrK C86S mutant induces a stronger inflammatory response at the early stage of infection, and displays reduced intestinal colonization and virulence at the later stage of infection, in comparison with the parent strain. Our results indicate that the pathogen V. parahaemolyticus perceives nitrite as a host-derived signal and responds by downregulating a proinflammatory factor (T3SS1), thus enhancing intestinal colonization and virulence.


Asunto(s)
Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidad , Anaerobiosis , Animales , Secuencia de Bases , Sitios de Unión , Células CACO-2 , Citocinas/metabolismo , Regulación hacia Abajo/genética , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Humanos , Modelos Biológicos , Nitratos/metabolismo , Nitritos/metabolismo , Nitrosación , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Conejos , Transcripción Genética , Vibrio parahaemolyticus/genética , Virulencia/genética
7.
Nat Commun ; 11(1): 5769, 2020 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-33188182

RESUMEN

Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.


Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Regulación de la Expresión Génica , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transcripción Genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Factor de Transcripción Activador 2/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Células HEK293 , Humanos , Luciferasas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fosforilación , Unión Proteica , Dedos de Zinc
8.
Nat Commun ; 11(1): 5762, 2020 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-33188184

RESUMEN

Occurrence of Colorectal cancer (CRC) is relevant with gut microbiota. However, role of IRF3, a key signaling mediator in innate immune sensing, has been barely investigated in CRC. Here, we unexpectedly found that the IRF3 deficient mice are hyper-susceptible to the development of intestinal tumor in AOM/DSS and Apcmin/+ models. Genetic ablation of IRF3 profoundly promotes the proliferation of intestinal epithelial cells via aberrantly activating Wnt signaling. Mechanically, IRF3 in resting state robustly associates with the active ß-catenin in the cytoplasm, thus preventing its nuclear translocation and cell proliferation, which can be relieved upon microbe-induced activation of IRF3. In accordance, the survival of CRC is clinically correlated with the expression level of IRF3. Therefore, our study identifies IRF3 as a negative regulator of the Wnt/ß-catenin pathway and a potential prognosis marker for Wnt-related tumorigenesis, and describes an intriguing link between gut microbiota and CRC via the IRF3-ß-catenin axis.


Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Núcleo Celular/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/prevención & control , Factor 3 Regulador del Interferón/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Enterocitos/metabolismo , Enterocitos/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Antígeno Ki-67/metabolismo , Ratones Endogámicos C57BL , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Análisis de Supervivencia , Vía de Señalización Wnt , beta Catenina/química
9.
Nat Commun ; 11(1): 5783, 2020 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-33188197

RESUMEN

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. However, current data analysis strategies use preset thresholds that can lead to suboptimal sensitivity/specificity tradeoffs and limited comparability across datasets. Here, we present a method based on statistical hypothesis testing on curves, which provides control of the false discovery rate. We apply it to several datasets probing epigenetic drugs and a metabolite. This leads us to detect off-target drug engagement, including the finding that the HDAC8 inhibitor PCI-34051 and its analog BRD-3811 bind to and inhibit leucine aminopeptidase 3. An implementation is available as an R package from Bioconductor ( https://bioconductor.org/packages/TPP2D ). We hope that our method will facilitate prioritizing targets from thermal profiling experiments.


Asunto(s)
Biología Computacional/métodos , Proteoma/metabolismo , Proteómica , Temperatura , Adenosina Trifosfato/metabolismo , Bases de Datos de Proteínas , Guanosina Trifosfato/metabolismo , Células Hep G2 , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Indoles/química , Indoles/farmacología , Leucil Aminopeptidasa/metabolismo , Ligandos , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Unión Proteica , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo
10.
Nat Commun ; 11(1): 5535, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-33139697

RESUMEN

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.


Asunto(s)
ADN Helicasas/genética , Reparación del ADN , Neoplasias/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Secuencia Conservada/genética , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Células HEK293 , Humanos , Mutación , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Unión Proteica/genética , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismo
11.
Nat Commun ; 11(1): 5506, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-33139728

RESUMEN

The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30-Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure-function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target proteins.


Asunto(s)
Acetiltransferasa C N-Terminal/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/enzimología , Acetilación , Secuencia de Aminoácidos , Cristalografía por Rayos X , Acetiltransferasa C N-Terminal/genética , Acetiltransferasa C N-Terminal/metabolismo , Naftoles , Unión Proteica , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Triazinas
12.
Nat Commun ; 11(1): 5588, 2020 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-33149112

RESUMEN

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.


Asunto(s)
Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/prevención & control , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Microscopía por Crioelectrón , Humanos , Pruebas de Neutralización , Unión Proteica , Conformación Proteica , Dominios Proteicos/inmunología , Receptores Virales/metabolismo
13.
Nat Commun ; 11(1): 5686, 2020 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-33173040

RESUMEN

Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H+/Fe2+ antiporter so that transport of each Fe2+ is coupled to transport of two H+ in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H+ and Fe2+. These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases.


Asunto(s)
Proteínas de Transporte de Catión/ultraestructura , Microscopía por Crioelectrón , Hepcidinas/metabolismo , Hierro/metabolismo , Anemia Ferropénica , Animales , Sitios de Unión , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/metabolismo , Humanos , Transporte Iónico , Unión Proteica
14.
Nat Commun ; 11(1): 5695, 2020 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-33173051

RESUMEN

Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called "activating adaptors" that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 µM. We propose that a tunable LIC1-adaptor interaction modulates dynein's motility in a cargo-specific manner.


Asunto(s)
Transporte Biológico/fisiología , Dineínas Citoplasmáticas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular , Cristalografía por Rayos X/métodos , Complejo Dinactina/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica
15.
Nat Commun ; 11(1): 5816, 2020 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-33199730

RESUMEN

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.


Asunto(s)
Cilios/metabolismo , Microcefalia/genética , Neurogénesis , Biogénesis de Organelos , Proteínas de Unión al ARN/genética , Ribosomas/metabolismo , Adulto , Animales , Secuencia de Bases , Encéfalo/embriología , Encéfalo/patología , Ciclo Celular , Nucléolo Celular/metabolismo , Centrosoma/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Ratones , Mutación/genética , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Pakistán , Linaje , Unión Proteica , Procesamiento Postranscripcional del ARN , ARN Ribosómico/genética , Proteínas de Unión al ARN/metabolismo , Pez Cebra/embriología
16.
J Mol Model ; 26(12): 341, 2020 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-33200284

RESUMEN

HER-2 type breast cancer is one of the most aggressive malignancies found in women. Tucatinib is recently developed and approved as a potential medicine to fight this disease. In this manuscript, we present the gross structural features of this compound and its reactivity and wave function properties using computational simulations. Density functional theory was used to optimise the ground state geometry of the molecule and molecular docking was used to predict biological activity. As the electrons interact with electromagnetic radiations, electronic excitations between different energy levels are analysed in detail using time-dependent density functional theory. Various intermolecular and intermolecular interactions are analysed and reaction sites for attacking electrophiles and nucleophiles identified. Information entropy calculations show that the compound is inherently stable. Docking with COVID-19 proteins show docking score of - 9.42, - 8.93, - 8.45 and - 8.32 kcal/mol respectively indicating high interaction between the drug and proteins. Hence, this is an ideal candidate to study repurposing of existing drugs to combat the pandemic.


Asunto(s)
Antineoplásicos/química , Antivirales/química , Betacoronavirus/química , Electrones , Oxazoles/química , Inhibidores de Proteasas/química , Piridinas/química , Quinazolinas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antineoplásicos/metabolismo , Antivirales/metabolismo , Betacoronavirus/enzimología , Sitios de Unión , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Reposicionamiento de Medicamentos , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Oxazoles/metabolismo , Inhibidores de Proteasas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Piridinas/metabolismo , Teoría Cuántica , Quinazolinas/metabolismo , Termodinámica , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo
17.
Cells ; 9(11)2020 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-33105869

RESUMEN

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease (COVID-19) pandemic, has so far resulted in more than 1.1 M deaths and 40 M cases worldwide with no confirmed remedy yet available. Since the first outbreak in Wuhan, China in December 2019, researchers across the globe have been in a race to develop therapies and vaccines against the disease. SARS-CoV-2, similar to other previously identified Coronaviridae family members, encodes several structural proteins, such as spike, envelope, membrane, and nucleocapsid, that are responsible for host penetration, binding, recycling, and pathogenesis. Structural biology has been a key player in understanding the viral infection mechanism and in developing intervention strategies against the new coronavirus. The spike glycoprotein has drawn considerable attention as a means to block viral entry owing to its interactions with the human angiotensin-converting enzyme 2 (ACE2), which acts as a receptor. Here, we review the current knowledge of SARS-CoV-2 and its interactions with ACE2 and antibodies. Structural information of SARS-CoV-2 spike glycoprotein and its complexes with ACE2 and antibodies can provide key input for the development of therapies and vaccines against the new coronavirus.


Asunto(s)
Betacoronavirus/química , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Humanos , Pandemias/prevención & control , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/prevención & control , Neumonía Viral/virología , Unión Proteica , Dominios Proteicos/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo
18.
Nat Commun ; 11(1): 5079, 2020 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-33033234

RESUMEN

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.


Asunto(s)
Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos , Matriz Extracelular/metabolismo , Ontología de Genes , Factores de Intercambio de Guanina Nucleótido/metabolismo , Folículo Piloso/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteína smad3/metabolismo , Transactivadores/metabolismo , Regulación hacia Arriba
19.
Nat Commun ; 11(1): 5063, 2020 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-33033242

RESUMEN

Genome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how coordination of ANP32E and H2A.Z contributes to genome-wide chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that ANP32E antagonizes H2A.Z accumulation to restrict chromatin accessibility genome-wide. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through subtle refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.


Asunto(s)
Cromatina/metabolismo , Genoma , Chaperonas Moleculares/metabolismo , Animales , Diferenciación Celular/genética , ADN Helicasas/metabolismo , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/metabolismo
20.
Nat Commun ; 11(1): 5066, 2020 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-33033255

RESUMEN

The inducible co-stimulator (ICOS) is a member of the CD28/B7 superfamily, and delivers a positive co-stimulatory signal to activated T cells upon binding to its ligand (ICOS-L). Dysregulation of this pathway has been implicated in autoimmune diseases and cancer, and is currently under clinical investigation as an immune checkpoint blockade. Here, we describe the molecular interactions of the ICOS/ICOS-L immune complex at 3.3 Å resolution. A central FDPPPF motif and residues within the CC' loop of ICOS are responsible for the specificity of the interaction with ICOS-L, with a distinct receptor binding orientation in comparison to other family members. Furthermore, our structure and binding data reveal that the ICOS N110 N-linked glycan participates in ICOS-L binding. In addition, we report crystal structures of ICOS and ICOS-L in complex with monoclonal antibodies under clinical evaluation in immunotherapy. Strikingly, antibody paratopes closely mimic receptor-ligand binding core interactions, in addition to contacting peripheral residues to confer high binding affinities. Our results uncover key molecular interactions of an immune complex central to human adaptive immunity and have direct implications for the ongoing development of therapeutic interventions targeting immune checkpoint receptors.


Asunto(s)
Anticuerpos/uso terapéutico , Complejo Antígeno-Anticuerpo/química , Ligando Coestimulador de Linfocitos T Inducibles/química , Proteína Coestimuladora de Linfocitos T Inducibles/química , Imitación Molecular/inmunología , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos CD28/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligando Coestimulador de Linfocitos T Inducibles/metabolismo , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Cinética , Ligandos , Modelos Moleculares , Unión Proteica , Multimerización de Proteína
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