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1.
Molecules ; 26(4)2021 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-33673080

RESUMEN

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-ß-cyclodextrin (methyl-ß-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.


Asunto(s)
Lípidos de la Membrana/química , Microdominios de Membrana/química , Receptores LHRH/química , Colesterol/química , Colesterol/genética , Espectroscopía de Resonancia por Spin del Electrón , Células HEK293 , Humanos , Lípidos de la Membrana/genética , Microdominios de Membrana/genética , Microdominios de Membrana/ultraestructura , Dominios Proteicos/genética , Receptores LHRH/genética , Receptores LHRH/uso terapéutico , Receptores LHRH/ultraestructura , Transducción de Señal/genética
2.
Molecules ; 26(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652554

RESUMEN

The translocator protein (TSPO) is a 18kDa transmembrane protein, ubiquitously present in human mitochondria. It is overexpressed in tumor cells and at the sites of neuroinflammation, thus representing an important biomarker, as well as a promising drug target. In mammalian TSPO, there are cholesterol-binding motifs, as well as a binding cavity able to accommodate different chemical compounds. Given the lack of structural information for the human protein, we built a model of human (h) TSPO in the apo state and in complex with PK11195, a molecule routinely used in positron emission tomography (PET) for imaging of neuroinflammatory sites. To better understand the interactions of PK11195 and cholesterol with this pharmacologically relevant protein, we ran molecular dynamics simulations of the apo and holo proteins embedded in a model membrane. We found that: (i) PK11195 stabilizes hTSPO structural fold; (ii) PK11195 might enter in the binding site through transmembrane helices I and II of hTSPO; (iii) PK11195 reduces the frequency of cholesterol binding to the lower, N-terminal part of hTSPO in the inner membrane leaflet, while this impact is less pronounced for the upper, C-terminal part in the outer membrane leaflet, where the ligand binding site is located; (iv) very interestingly, cholesterol most frequently binds simultaneously to the so-called CRAC and CARC regions in TM V in the free form (residues L150-X-Y152-X(3)-R156 and R135-X(2)-Y138-X(2)-L141, respectively). However, when the protein is in complex with PK11195, cholesterol binds equally frequently to the CRAC-resembling motif that we observed in TM I (residues L17-X(2)-F20-X(3)-R24) and to CRAC in TM V. We expect that the CRAC-like motif in TM I will be of interest in future experimental investigations. Thus, our MD simulations provide insight into the structural features of hTSPO and the previously unknown interplay between PK11195 and cholesterol interactions with this pharmacologically relevant protein.


Asunto(s)
Colesterol/química , Isoquinolinas/química , Estructura Secundaria de Proteína , Receptores de GABA/ultraestructura , Sitios de Unión/genética , Transporte Biológico/genética , Humanos , Ligandos , Mitocondrias/genética , Mitocondrias/ultraestructura , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica/genética , Dominios Proteicos/genética , Pliegue de Proteína , Receptores de GABA/química
3.
PLoS One ; 16(3): e0246981, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33730017

RESUMEN

Nidoviruses and arenaviruses are the only known RNA viruses encoding a 3'-5' exonuclease domain (ExoN). The proofreading activity of the ExoN domain has played a key role in the growth of nidoviral genomes, while in arenaviruses this domain partakes in the suppression of the host innate immune signaling. Sequence and structural homology analyses suggest that these proteins have been hijacked from cellular hosts many times. Analysis of the available nidoviral ExoN sequences reveals a high conservation level comparable to that of the viral RNA-dependent RNA polymerases (RdRp), which are the most conserved viral proteins. Two highly preserved zinc fingers are present in all nidoviral exonucleases, while in the arenaviral protein only one zinc finger can be identified. This is in sharp contrast with the reported lack of zinc fingers in cellular ExoNs, and opens the possibility of therapeutic strategies in the struggle against COVID-19.


Asunto(s)
Exonucleasas/genética , Dominios Proteicos/genética , ARN Viral/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Arenavirus/genética , Humanos , Inmunidad Innata/genética , Nidovirales/genética , Virus ARN/genética , /genética , Dedos de Zinc/genética
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): 1176, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33608534

RESUMEN

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.


Asunto(s)
Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cisteína/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/genética
6.
Nat Commun ; 12(1): 585, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33500419

RESUMEN

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.


Asunto(s)
Síndrome de Bloom/genética , Replicación del ADN , ADN de Cadena Simple/metabolismo , RecQ Helicasas/metabolismo , Proteína de Replicación A/metabolismo , Secuencias de Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Daño del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Mitosis/genética , Mutación , Unión Proteica/genética , Dominios Proteicos/genética , RecQ Helicasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparación del ADN por Recombinación/genética
7.
Nat Commun ; 12(1): 704, 2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33514736

RESUMEN

p53 mutations with single amino acid changes in cancer often lead to dominant oncogenic changes. Here, we have developed a mouse model of gain-of-function (GOF) p53-driven lung cancer utilizing conditionally active LSL p53-R172H and LSL K-Ras-G12D knock-in alleles that can be activated by Cre in lung club cells. Mutation of the p53 transactivation domain (TAD) (p53-L25Q/W26S/R172H) eliminating significant transactivation activity resulted in loss of tumorigenicity, demonstrating that transactivation mediated by or dependent on TAD is required for oncogenicity by GOF p53. GOF p53 TAD mutations significantly reduce phosphorylation of nearby p53 serine 20 (S20), which is a target for PLK3 phosphorylation. Knocking out PLK3 attenuated S20 phosphorylation along with transactivation and oncogenicity by GOF p53, indicating that GOF p53 exploits PLK3 to trigger its transactivation capability and exert oncogenic functions. Our data show a mechanistic involvement of PLK3 in mutant p53 pathway of oncogenesis.


Asunto(s)
Carcinogénesis/genética , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Mutación con Ganancia de Función , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Fosforilación/genética , Dominios Proteicos/genética , Proteínas Serina-Treonina Quinasas/genética , Serina/metabolismo , Esferoides Celulares , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo
8.
Plant Physiol Biochem ; 159: 372-382, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33444896

RESUMEN

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.


Asunto(s)
Arabidopsis , Rubia , Tolerancia a la Sal , Termotolerancia , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultivo de Célula , Regulación de la Expresión Génica de las Plantas , Oxidorreductasas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Dominios Proteicos/genética , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rubia/genética , Rubia/metabolismo , Tolerancia a la Sal/genética , Estrés Fisiológico/genética , Termotolerancia/genética
9.
Nat Commun ; 12(1): 384, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33452263

RESUMEN

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8-SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Difosfatos/metabolismo , Regulación de la Expresión Génica de las Plantas , Fosfatos de Inositol/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/ultraestructura , Cristalografía por Rayos X , Mutación , Proteínas Nucleares/genética , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/ultraestructura
10.
Arch Virol ; 166(3): 767-778, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33420816

RESUMEN

Complement component 1 Q subcomponent-binding protein (C1QBP) has been shown to interact with the porcine circovirus type 2 (PCV2) Cap protein. Here, using yeast two-hybrid (Y2H) and co-immunoprecipitation assays, as well as laser confocal microscopy, the interaction between C1QBP and Cap was confirmed. Furthermore, overexpression of C1QBP in cells altered the intracellular location of Cap, which was observed using confocal microscopy and verified by detection of Cap in nuclear protein extracts in a Western blot assay. By inhibiting nuclear transport of Cap, overexpression of C1QBP downregulated PCV2 proliferation in PK-15 cells, as determined by quantitative polymerase chain reaction (qPCR). As C1QBP plays a similar role in a fusion of green fluorescent protein (GFP) with the Cap nuclear localisation signal (NLS) sequence, (CapNLS-GFP), we propose that the target site for C1QBP in Cap is possibly located in the NLS region. Considering all the results together, this study demonstrated that C1QBP interacts with the Cap NLS region, resulting in changes in the intracellular localisation of the Cap protein. We confirmed that overexpression of C1QBP inhibits the proliferation of PCV2, and this is possibly related to the function of C1QBP in controlling nuclear transport of Cap.


Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de la Cápside/metabolismo , Circovirus/crecimiento & desarrollo , Complemento C1q/metabolismo , Replicación Viral/fisiología , Animales , Línea Celular , Chlorocebus aethiops , ADN Viral/metabolismo , Células HEK293 , Humanos , Dominios Proteicos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Porcinos , Células Vero
11.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-33477809

RESUMEN

Proinflammatory response and mitochondrial dysfunction are related to the pathogenesis of neurodegenerative diseases (NDs). Nuclear factor κB (NFκB) activation has been shown to exaggerate proinflammation and mitochondrial dysfunction, which underlies NDs. CDGSH iron-sulfur domain 2 (CISD2) has been shown to be associated with peroxisome proliferator-activated receptor-ß (PPAR-ß) to compete for NFκB and antagonize the two aforementioned NFκB-provoked pathogeneses. Therefore, CISD2-based strategies hold promise in the treatment of NDs. CISD2 protein belongs to the human NEET protein family and is encoded by the CISD2 gene (located at 4q24 in humans). In CISD2, the [2Fe-2S] cluster, through coordinates of 3-cysteine-1-histidine on the CDGSH domain, acts as a homeostasis regulator under environmental stress through the transfer of electrons or iron-sulfur clusters. Here, we have summarized the features of CISD2 in genetics and clinics, briefly outlined the role of CISD2 as a key physiological regulator, and presented modalities to increase CISD2 activity, including biomedical engineering or pharmacological management. Strategies to increase CISD2 activity can be beneficial for the prevention of inflammation and mitochondrial dysfunction, and thus, they can be applied in the management of NDs.


Asunto(s)
Proteínas de la Membrana/genética , FN-kappa B/genética , Enfermedades Neurodegenerativas/tratamiento farmacológico , PPAR-beta/genética , Cisteína/genética , Histidina/genética , Homeostasis/genética , Humanos , Hierro/metabolismo , Proteínas con Hierro-Azufre/genética , Proteínas de la Membrana/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Dominios Proteicos/genética
12.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118887, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33075383

RESUMEN

The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers.


Asunto(s)
Dimerización , Conformación Proteica , Receptores Acoplados a Proteínas G/ultraestructura , Proteínas Recombinantes de Fusión/genética , Survivin/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Secuencias de Aminoácidos/genética , Membrana Celular/química , Proteínas de Unión al GTP/química , Células HEK293 , Humanos , Modelos Moleculares , Dominios Proteicos/genética , Multimerización de Proteína/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Survivin/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química
13.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118894, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33091504

RESUMEN

HIPK2 is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in a broad spectrum of biological processes. We have previously reported that the expression of HIPK2 is auto-regulated by a mechanism that depends on the activity of its kinase domain, leading to decreased expression of kinase-dead versus wild-type HIPK2. We have now explored this mechanism in more detail. Differential expression of wild-type and kinase-dead HIPK2 is dependent on sequences located in the C-terminal part of HIPK2, but is only observed when this part of HIPK2 is translated together with the defective kinase domain. On their own, both the defective kinase domain and the C-terminal amino acid sequences are expressed at normal levels and independently of kinase activity. Insertion of a 2A-ribosomal skipping sequence into the HIPK2 coding sequence revealed that the differential expression of wild-type and kinase-dead HIPK2 is caused by degradation of nascent kinase-dead HIPK2. Because HIPK2 is constitutively active and auto-activates its kinase domain already during its translation we speculate that the regulatory mechanism discovered here serves as a quality control mechanism that leads to degradation of nascent kinase molecules with defective kinase domains. Overall our work provides insight into a novel auto-regulatory mechanism of HIPK2 expression, thereby adding a new layer of control to the regulation of HIPK2.


Asunto(s)
Proteínas Portadoras/genética , Fosforilación/genética , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Secuencia de Aminoácidos/genética , Proteínas Portadoras/química , Regulación de la Expresión Génica/genética , Células HeLa , Células Hep G2 , Humanos , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Serina-Treonina Quinasas/química
14.
Ann Neurol ; 89(3): 573-586, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33325057

RESUMEN

OBJECTIVE: We aimed to characterize the phenotypic spectrum and functional consequences associated with variants in the gene GABRB2, coding for the γ-aminobutyric acid type A (GABAA ) receptor subunit ß2. METHODS: We recruited and systematically evaluated 25 individuals with variants in GABRB2, 17 of whom are newly described and 8 previously reported with additional clinical data. Functional analysis was performed using a Xenopus laevis oocyte model system. RESULTS: Our cohort of 25 individuals from 22 families with variants in GABRB2 demonstrated a range of epilepsy phenotypes from genetic generalized epilepsy to developmental and epileptic encephalopathy. Fifty-eight percent of individuals had pharmacoresistant epilepsy; response to medications targeting the GABAergic pathway was inconsistent. Developmental disability (present in 84%) ranged from mild intellectual disability to severe global disability; movement disorders (present in 44%) included choreoathetosis, dystonia, and ataxia. Disease-associated variants cluster in the extracellular N-terminus and transmembrane domains 1-3, with more severe phenotypes seen in association with variants in transmembrane domains 1 and 2 and the allosteric binding site between transmembrane domains 2 and 3. Functional analysis of 4 variants in transmembrane domains 1 or 2 (p.Ile246Thr, p.Pro252Leu, p.Ile288Ser, p.Val282Ala) revealed strongly reduced amplitudes of GABA-evoked anionic currents. INTERPRETATION: GABRB2-related epilepsy ranges broadly in severity from genetic generalized epilepsy to developmental and epileptic encephalopathies. Developmental disability and movement disorder are key features. The phenotypic spectrum is comparable to other GABAA receptor-encoding genes. Phenotypic severity varies by protein domain. Experimental evidence supports loss of GABAergic inhibition as the mechanism underlying GABRB2-associated neurodevelopmental disorders. ANN NEUROL 2021;89:573-586.


Asunto(s)
Epilepsia/fisiopatología , Trastornos del Movimiento/fisiopatología , Trastornos del Neurodesarrollo/fisiopatología , Receptores de GABA-A/genética , Adolescente , Adulto , Animales , Ataxia/genética , Ataxia/fisiopatología , Atetosis/genética , Atetosis/fisiopatología , Niño , Preescolar , Corea/genética , Corea/fisiopatología , Estudios de Cohortes , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/fisiopatología , Epilepsia Refractaria/genética , Epilepsia Refractaria/fisiopatología , Distonía/genética , Distonía/fisiopatología , Epilepsia/genética , Femenino , Genotipo , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/fisiopatología , Masculino , Persona de Mediana Edad , Trastornos del Movimiento/genética , Mutación Missense , Trastornos del Neurodesarrollo/genética , Oocitos , Técnicas de Placa-Clamp , Fenotipo , Dominios Proteicos/genética , Xenopus laevis , Adulto Joven
15.
Gene ; 764: 145081, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-32860897

RESUMEN

Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. In this study, a conserved domain RPOLA-N of the RPA190 gene of P. capsici (RPA190-pc) was identified from the P. capsici SD1-9 strain. The role of the RPA190-pc underlying the metalaxyl resistance of P. capsici was investigated. Three P. capsici mutants, two with downregulated RPA190-pc (SD1-9C-3 and C-4) expression and one showing upregulation (OESD1-9-1), were obtained by Polyethylene Glycol (PEG) mediated protoplast transformations of P. capsici SD1-9. Quantitative real-time reverse transcription PCR results showed that RPA190-pc was downregulated by more than 60% in SD1-9C-3/C-4 and upregulated 3-fold in OESD1-9-1 compared with that of the control strain SD1-9. Evaluation of the metalaxyl resistance of these three transformants showed that the EC50 values of metalaxyl against SD1-9C-3, SD1-9C-4, and OESD1-9-1 were 120.0 µg·mL-1, 24.4 µg·mL-1, and 15573.0 µg·mL-1, respectively, corresponding to 63.3% decrease, 92.5% decrease, and 47.7-fold increase relative to the EC50 value in SD1-9. Compared with SD1-9, the mycelia of transformants SD1-9C-3, SD1-9C-4, and OESD1-9-1 showed more branches and shorter branches; and the transformants had different pathogenicity to different hosts plants. The expression of the candidate gene RPA190-pc during 10 life-history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage, and it gradually increased with the increase of SD1 and SD1-9 infection time of pepper leaves, indicated that RPA190-pc may be related to the growth and pathogenicity of P. capsici. These results indicate that the expression of RPA190-pc is involved in the regulation of P. capsici resistance to metalaxyl.


Asunto(s)
Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Fungicidas Industriales/farmacología , Phytophthora/genética , ARN Polimerasa I/genética , Alanina/análogos & derivados , Alanina/farmacología , Capsicum/microbiología , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Estadios del Ciclo de Vida/genética , Mutación , Micelio/genética , Micelio/crecimiento & desarrollo , Phytophthora/efectos de los fármacos , Phytophthora/crecimiento & desarrollo , Phytophthora/patogenicidad , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Hojas de la Planta/microbiología , Dominios Proteicos/genética , Esporangios/genética , Esporangios/crecimiento & desarrollo , Virulencia/genética
16.
Gene ; 764: 145094, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-32860898

RESUMEN

Long chain acyl-CoA synthetases (ACSLs), which drive the conversion of long chain fatty acid into acyl-CoA, an ingredient of lipid synthesis, have been well-acknowledged to exert an indispensable role in many metabolic processes in mammals, especially lipid metabolism. However, in chicken, the evolutionary characteristics, expression profiles and regulatory mechanisms of ACSL gene family are rarely understood. Here, we analyzed the genomic synteny, gene structure, evolutionary event and functional domains of the ACSL gene family members using bioinformatics methods. The spatiotemporal expression profiles of ACSL gene family, and their regulatory mechanism were investigated via bioinformatics analysis incorporated with in vivo and in vitro estrogen-treated experiments. Our results indicated that ACSL2 gene was indeed evolutionarily lost in the genome of chicken. Chicken ACSLs shared an AMP-binding functional domain, as well as highly conversed ATP/AMP and FACS signature motifs, and were clustered into two clades, ACSL1/5/6 and ACSL3/4, based on high sequence similarity, similar gene features and conversed motifs. Chicken ACSLs showed differential tissue expression distributions, wherein the significantly decreased expression level of ACSL1 and the significantly increased expression level of ACSL5 were found, respectively, the expression levels of the other ACSL members remained unchanged in the liver of peak-laying hens versus pre-laying hens. Moreover, the transcription activity of ACSL1, ACSL3 and ACSL4 was silenced and ACSL6 was activated by estrogen, but no response to ACSL5. In conclusion, though having highly conversed functional domains, chicken ACSL gene family is organized into two separate groups, ACSL1/5/6 and ACSL3/4, and exhibits varying expression profiles and estrogen effects. These results not only pave the way for better understanding the specific functions of ACSL genes in avian lipid metabolism, but also provide a valuable evidence for gene family characteristics.


Asunto(s)
Pollos/genética , Coenzima A Ligasas/genética , Evolución Molecular , Metabolismo de los Lípidos/genética , Familia de Multigenes/genética , Acilcoenzima A/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Pollos/crecimiento & desarrollo , Pollos/metabolismo , Coenzima A Ligasas/metabolismo , Biología Computacional , Estrógenos/metabolismo , Ácidos Grasos/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hepatocitos , Cultivo Primario de Células , Dominios Proteicos/genética , Análisis Espacio-Temporal , Sintenía
17.
Thromb Haemost ; 120(12): 1700-1715, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33368089

RESUMEN

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.


Asunto(s)
Anticoagulantes/farmacología , Antivirales/farmacología , Enoxaparina/farmacología , Heparina/farmacología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Anticoagulantes/uso terapéutico , Antivirales/uso terapéutico , Chlorocebus aethiops , Enoxaparina/uso terapéutico , Heparina/uso terapéutico , Humanos , Simulación de Dinámica Molecular , Nebulizadores y Vaporizadores , Unión Proteica , Conformación Proteica , Dominios Proteicos/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Relación Estructura-Actividad , Células Vero , Internalización del Virus
18.
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
19.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-33004795

RESUMEN

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Asunto(s)
Antígenos de Neoplasias/ultraestructura , Proteínas de Neoplasias/ultraestructura , Neoplasias/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencias de Aminoácidos , Aminoquinolinas/farmacología , Aminoquinolinas/uso terapéutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinogénesis/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Mutagénesis , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Prueba de Estudio Conceptual , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Dominios Proteicos/genética , Mapeo de Interacción de Proteínas , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/genética , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética
20.
J Transl Med ; 18(1): 329, 2020 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-32867854

RESUMEN

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.


Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Eliminación de Secuencia , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Betacoronavirus/patogenicidad , Enfermedades Transmisibles Emergentes/virología , Infecciones por Coronavirus/epidemiología , Frecuencia de los Genes , Genoma Viral , Geografía , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estadística & datos numéricos , Fenilalanina/genética , Neumonía Viral/epidemiología , Dominios Proteicos/genética , Serina/genética , Proteínas no Estructurales Virales/química , Virulencia/genética , Replicación Viral/genética
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