Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.444
Filtrar
1.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024075
2.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: covidwho-834865
3.
Nat Commun ; 11(1): 4370, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873792

RESUMO

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sítio Alostérico/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Sistema de Sinalização das MAP Quinases/genética , Simulação de Acoplamento Molecular , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/ultraestrutura , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
4.
Nat Commun ; 11(1): 4506, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908149

RESUMO

Bacteriophages play critical roles in the biosphere, but their vast genomic diversity has obscured their evolutionary origins, and phylogenetic analyses have traditionally been hindered by their lack of universal phylogenetic marker genes. In this study we mine metagenomic data and identify a clade of Caudovirales that encodes the ß and ß' subunits of multi-subunit RNA polymerase (RNAP), a high-resolution phylogenetic marker which enables detailed evolutionary analyses. Our RNAP phylogeny revealed that the Caudovirales RNAP forms a clade distinct from cellular homologs, suggesting an ancient acquisition of this enzyme. Within these multimeric RNAP-encoding Caudovirales (mReC), we find that the similarity of major capsid proteins and terminase large subunits further suggests they form a distinct clade with common evolutionary origin. Our study characterizes a clade of RNAP-encoding Caudovirales and suggests the ancient origin of this enzyme in this group, underscoring the important role of viruses in the early evolution of life on Earth.


Assuntos
Evolução Biológica , Caudovirales/genética , RNA Polimerases Dirigidas por DNA/genética , Subunidades Proteicas/genética , Proteínas Virais/genética , DNA Viral/genética , Conjuntos de Dados como Assunto , Transferência Genética Horizontal , Metagenômica , Filogenia , Análise de Sequência de DNA
5.
PLoS Biol ; 18(8): e3000826, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776935

RESUMO

Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and ßCaMKII-deficient (αßCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αßCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αßCaMKII DKO neurons was restored by active ßCaMKII but not inactive ßCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to ßCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.


Assuntos
Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/genética , Animais , Astrócitos/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Fosforilação , Cultura Primária de Células , Subunidades Proteicas/deficiência , Sinapses/fisiologia , Transmissão Sináptica , Imagem com Lapso de Tempo
6.
PLoS One ; 15(8): e0237711, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810182

RESUMO

Alleles at the Glu-1 loci play important roles in the functional properties of wheat flour. The effects of various high-molecular-weight glutenin subunit (HMW-GS) compositions on quality traits and bread-making properties were evaluated using 235 doubled haploid lines (DHs). The experiment was conducted in a split plot design with two water regimes as the main plot treatment, and DH lines as the subplot treatments. Results showed that the presence of subunit pair 5+10 at the Glu-D1 locus, either alone or in combination with others, appears to provide an improvement in quality and bread-making properties. At the Glu-A1 locus, subunit 1 produced a higher Zeleny sedimentation value (Zel) and stretch area (SA) than subunit 2* when subunits 14+15 and 5+10 were expressed at the Glu-B1 and Glu-D1 loci, and 2* had a positive effect on the maximum dough resistance (Rmax) when subunits 14+15 and 5'+12 were expressed at the Glu-B1 and Glu-D1 loci, respectively. Given subunit 1 at the Glu-A1 locus and 5'+12 at the Glu-D1 locus, the effects of Glu-B1 subunits 14+15 on the tractility (Tra), dough stability time (ST), and dough development time (DT) under the well-watered regime were significantly higher than those of Glu-B1 subunits 13+16. However, 13+16 had a positive effect on SA under the rain-fed regime when subunits 2* and 5+10 were expressed at the Glu-A1 and Glu-D1 loci, respectively. Multiple comparisons analysis revealed that the Zel and Rmax of the six subunits and eight HMW-GS compositions were stable under different water regimes. Overall, subunit compositions 1, 13+16 and 5+10 and 1, 14+15 and 5+10 had higher values for quality traits and bread-baking properties under the two water regimes. These results could play a positive guiding role in selecting and popularizing varieties suitable for production and cultivation in local areas.


Assuntos
Qualidade dos Alimentos , Glutens/genética , Melhoramento Vegetal , Subunidades Proteicas/genética , Triticum/química , Irrigação Agrícola/métodos , Pão/normas , China , Farinha/normas , Genes de Plantas/genética , Loci Gênicos , Glutens/metabolismo , Haploidia , Peso Molecular , Subunidades Proteicas/química , Triticum/genética , Triticum/crescimento & desenvolvimento
7.
Eur J Endocrinol ; 183(4): 369-379, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32621582

RESUMO

Background: The '3PAs' syndrome, associating pituitary adenoma (PA) and pheochromocytoma/paraganglioma (PPGL), is sometimes associated with mutations in PPGL-predisposing genes, such as SDHx or MAX. In '3PAs' syndrome, PAs can occur before PPGL, suggesting a new gateway into SDHx/MAX-related diseases. Objective: To determine the SDHx/MAX mutation prevalence in patients with isolated PAs and characterize PAs of patients with SDHx/MAX mutations. Design: Genes involved in PAs (AIP/MEN1/CDKN1B) or PPGLs (SDHx/MAX) were sequenced in patients with isolated PAs. We then conducted a review of cases of PA in the setting of '3PAs' syndrome. Results: A total of 263 patients were recruited. Seven (likely) pathogenic variants were found in AIP, two in MEN1, two in SDHA, and one in SDHC. The prevalence of SDHx mutations reached 1.1% (3/263). Of 31 reported patients with PAs harboring SDHx/MAX mutations (28 published cases and 3 cases reported here), 6/31 (19%) developed PA before PPGL and 8/31 (26%) had isolated PA. The age of onset was later than in patients with AIP/MEN1 mutations. PAs were mainly macroprolactinomas and showed intracytoplasmic vacuoles seen on histopathology. Conclusions: We discovered SDHx mutations in patients bearing PA who had no familial or personal history of PPGL. However, the question of incidental association remains unresolved and data to determine the benefit of SDHx/MAX screening in these patients are lacking. We recommend that patients with isolated PA should be carefully examined for a family history of PPGLs. A family history of PPGL, as well as the presence of intracytoplasmic vacuoles in PA, requires SDHx/MAX genetic testing of patients.


Assuntos
Adenoma/genética , Mutação em Linhagem Germinativa , Neoplasias Hipofisárias/genética , Succinato Desidrogenase/genética , Adenoma/epidemiologia , Adenoma/patologia , Adolescente , Neoplasias das Glândulas Suprarrenais/epidemiologia , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idade de Início , Idoso , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Criança , Análise Mutacional de DNA/métodos , Feminino , França/epidemiologia , Predisposição Genética para Doença , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Paraganglioma/epidemiologia , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/epidemiologia , Feocromocitoma/genética , Feocromocitoma/patologia , Neoplasias Hipofisárias/epidemiologia , Neoplasias Hipofisárias/patologia , Prolactinoma/epidemiologia , Prolactinoma/genética , Prolactinoma/patologia , Subunidades Proteicas/genética , Estudos Retrospectivos , Adulto Jovem
8.
Proc Natl Acad Sci U S A ; 117(28): 16283-16291, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32611810

RESUMO

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.


Assuntos
Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Abelhas/metabolismo , Relação Dose-Resposta a Droga , Drosophila melanogaster/metabolismo , Proteínas de Insetos/agonistas , Proteínas de Insetos/genética , Oócitos/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Xenopus laevis
9.
Nat Commun ; 11(1): 3583, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681005

RESUMO

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.


Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Engenharia de Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato
10.
Proc Natl Acad Sci U S A ; 117(27): 15702-15711, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576691

RESUMO

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIß). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.


Assuntos
Cerebelo/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Neurônios/metabolismo , RNA Polimerase III/genética , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Cerebelo/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Camundongos Knockout , Neurônios/patologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , Subunidades Proteicas/genética
11.
PLoS One ; 15(6): e0234080, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479525

RESUMO

In the present study we have characterized the biophysical properties of wild-type (WT) α1ß2 and α3ß2 GABAA receptors and probed the molecular basis for the observed differences. The activation and desensitization behavior and the residual currents of the receptors expressed in HEK293 cells were determined in whole-cell patch clamp recordings. Kinetic parameters of α1ß2 and α3ß2 activation differed significantly, with α1ß2 and α3ß2 exhibiting rise times (10-90%) of 24 ± 2 ms and 51 ± 7 ms, respectively. In contrast, the two receptors exhibited largely comparable desensitization behavior with decay currents that could be fitted to exponential functions with two or three components. Most notably, the two receptor compositions displayed different degrees of desentization, with the residual currents of α1ß2 and α3ß2 constituting 34 ± 2% and 21 ± 2% of the peak current, respectively. The respective contributions of the extracellular domains and the transmembrane/intracellular domains of the α-subunit to these physiological profiles were next assessed in recordings from cells expressing αß2 receptors comprising chimeric α-subunits. The rise times displayed by α1ECD/α3TMDß2 and α3ECD/α1TMDß2 receptors were intermediate to those of WT α1ß2 and WT α3ß2, and the distribution of the different components of the current decays exhibited by the two chimeric receptors followed the same pattern as the two WT receptors. The residual current exhibited by α1ECD/α3TMDß2 (23 ± 3%) was similar to that of α3ß2 but significantly different from that of α1ß2, whereas the residual current displayed by α3ECD/α1TMDß2 (27 ± 2%) was intermediate to and did not differ significantly from either of the WT receptors. This points to molecular differences in the transmembrane/intracellular domains of the α-subunit as the main determinants of the observed differences in receptor physiology between α1ß2 and α3ß2 receptors.


Assuntos
Receptores de GABA-A/metabolismo , Potenciais de Ação/efeitos dos fármacos , Células HEK293 , Humanos , Rim/fisiologia , Cinética , Técnicas de Patch-Clamp , Domínios Proteicos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/genética , Ácido gama-Aminobutírico/farmacologia
12.
Clin Sci (Lond) ; 134(12): 1457-1472, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32514535

RESUMO

The chromatin remodeling complex SWI/SNF regulates the accessibility of target genes to transcription factors and plays a critical role in the tumorigenesis of hepatocellular carcinoma (HCC). The SWI/SNF complex is assembled from approximately 15 subunits, and most of these subunits have distinct roles and are often aberrantly expressed in HCC. A comprehensive exploration of the expression and clinical significance of these subunits would be of great value. In the present study, we obtained the gene expression profile of each SWI/SNF subunit and the corresponding clinical information from The Cancer Genome Atlas (TCGA). We found that 14 out of the 15 SWI/SNF subunits were significantly increased in HCC tissues compared with paired normal liver tissues, and 11 subunits were significantly associated with overall survival (OS). We identified a four-gene prognostic signature including actin-like 6A (ACTL6A), AT-rich interaction domain 1A (ARID1A), SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily C member 1 (SMARCC1) and SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily D, member 1 (SMARCD1) that could effectively predict OS in HCC patients. Among the genes, SMARCD1 has the most prognostic value. We further conducted in vitro and in vivo experiments and revealed that SMARCD1 promotes liver cancer growth by activating the mTOR signaling pathway. In conclusion, our study has revealed that the expression of SWI/SNF complex subunits, especially SMARCD1, is highly associated with HCC development and acts as a promising prognostic predictor.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Subunidades Proteicas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Cromossômicas não Histona/genética , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Subunidades Proteicas/genética , Transdução de Sinais , Resultado do Tratamento
13.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
14.
PLoS Genet ; 16(5): e1008681, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463832

RESUMO

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Transposases/fisiologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico/genética , Células Cultivadas , Domesticação , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Proteínas do Grupo Polycomb/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células Sf9 , Spodoptera , Transposases/genética
15.
Nat Commun ; 11(1): 1643, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242014

RESUMO

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Animais , Complexo I de Transporte de Elétrons/genética , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
16.
PLoS Genet ; 16(3): e1008625, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176688

RESUMO

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and ß4 subunits. ß4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat ß4b cDNA. Heterologously expressed wild-type ß4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the ß4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, ß4b and ß4b-L125P augmented the calcium current amplitudes, however, ß4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of ß4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype ß4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of ß4b in cultured myotubes and hippocampal neurons. While binding of ß4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between ß4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of ß4b.


Assuntos
Canais de Cálcio/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Subunidades Proteicas/genética , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo N/genética , Células Cultivadas , Feminino , Regulação da Expressão Gênica/genética , Células HEK293 , Hipocampo/fisiologia , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos BALB C , Neurônios/metabolismo , Terminações Pré-Sinápticas/fisiologia , Isoformas de Proteínas/genética , Ratos , Transmissão Sináptica/genética
17.
Nat Struct Mol Biol ; 27(3): 288-296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123390

RESUMO

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.


Assuntos
ADP Ribose Transferases/química , Antígenos de Bactérias/química , Toxinas Bacterianas/química , Clostridium perfringens/química , Subunidades Proteicas/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Clonagem Molecular , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
18.
Mol Cell Biol ; 40(11)2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32179551

RESUMO

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 at lysine 27 (H3K27) in genomic regions of most eukaryotes and is critical for maintenance of the associated transcriptional repression. However, the mechanisms that shape the distribution of H3K27 methylation, such as recruitment of PRC2 to chromatin and/or stimulation of PRC2 activity, are unclear. Here, using a forward genetic approach in the model organism Neurospora crassa, we identified two alleles of a gene, NCU04278, encoding an unknown PRC2 accessory subunit (PAS). Loss of PAS resulted in losses of H3K27 methylation concentrated near the chromosome ends and derepression of a subset of associated subtelomeric genes. Immunoprecipitation followed by mass spectrometry confirmed reciprocal interactions between PAS and known PRC2 subunits, and sequence similarity searches demonstrated that PAS is not unique to N. crassa PAS homologs likely influence the distribution of H3K27 methylation and underlying gene repression in a variety of fungal lineages.


Assuntos
Histonas/metabolismo , Neurospora crassa/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas de Protozoários/metabolismo , Deleção de Genes , Lisina/metabolismo , Metilação , Neurospora crassa/genética , Complexo Repressor Polycomb 2/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/genética
19.
BMC Med Genet ; 21(1): 51, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171285

RESUMO

INTRODUCTION: The AP4B1 gene encodes a subunit of adaptor protein complex-4 (AP4), a component of intracellular transportation of proteins which plays important roles in neurons. Bi-allelic mutations in AP4B1 cause autosomal recessive spastic paraplegia-47(SPG47). CASE PRESENTATION: Here we present a Chinese patient with spastic tetraplegia, moderate psychomotor development delay and febrile seizures plus. Brain MRIs showed dilated supratentorial ventricle, thin posterior and splenium part of corpus callosum. The patient had little progress through medical treatments and rehabilitating regimens. Whole exome sequencing identified novel compound heterozygous truncating variants c.1207C > T (p.Gln403*) and c.52_53delAC (p.Cys18Glnfs*7) in AP4B1 gene. Causal mutations in AP4B1 have been reported in 29 individuals from 22 families so far, most of which are homozygous mutations. CONCLUSIONS: Our study enriched the genetic and phenotypic spectrum of SPG47. Early discovery, diagnosis and proper treatment on the conditions generally increase chances of improvement on the quality of life for patients.


Assuntos
Complexo 4 de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Proteínas de Ligação a DNA/genética , Transtornos Psicomotores/genética , Quadriplegia/genética , Proteínas de Ligação a RNA/genética , Convulsões Febris/genética , Grupo com Ancestrais do Continente Asiático , Criança , China , Códon sem Sentido , Heterozigoto , Humanos , Masculino , Fenótipo , Subunidades Proteicas/genética , Transtornos Psicomotores/complicações , Quadriplegia/complicações , Convulsões Febris/complicações , Sequenciamento Completo do Exoma
20.
Zoolog Sci ; 37(1): 7-13, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32068369

RESUMO

The outer dynein arm-docking complex (ODA-DC), which was first identified in the green alga Chlamydomonas reinhardtii, is a protein complex that mediates the binding of axonemal dynein and doublet microtubules. To gain a better understanding of the evolutionary conservation and functional diversity of the ODA-DC, we knocked down a homolog of DC2, a major subunit of the ODA-DC, in the planarian Schmidtea mediterranea. Planaria are carnivorous flatworms that move by beating cilia on their ventral surface against a secreted mucus layer. These organisms have recently been used for cilia research because knockdown of flatworm genes by RNA interference (RNAi) is readily achieved through feeding with double-stranded RNA (dsRNA). Lack of DC2 in S. mediterranea caused several defects in cilia, including low beat frequency, decreased ciliary density, and a reduction in ciliary length. The loss of DC2 function C. reinhardtii mutant oda1 shows slow jerky swimming, but has two flagella of almost normal length. These data suggest that the function of a DC2 homolog in S. mediterranea cilia may be somewhat different from DC2 in C. reinhardtii flagella.


Assuntos
Dineínas do Axonema/metabolismo , Cílios/patologia , Planárias/metabolismo , Sequência de Aminoácidos , Animais , Dineínas do Axonema/genética , Cílios/genética , Cílios/metabolismo , Cílios/ultraestrutura , Flagelos , Microscopia Eletrônica de Transmissão , Movimento , Planárias/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA