Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.168
Filtrar
1.
Science ; 373(6552): 306-315, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34437148

RESUMO

Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/química , Código das Histonas , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fatores de Transcrição/química
2.
Nat Commun ; 12(1): 4900, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385433

RESUMO

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.


Assuntos
Anquirinas/metabolismo , Mitocôndrias Musculares/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Musculares/metabolismo , Sarcolema/metabolismo , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia
3.
Nat Commun ; 12(1): 4843, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376693

RESUMO

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Repetições de Microssatélites/genética , Proteínas de Ligação a Telômeros/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Reparo do DNA por Junção de Extremidades , DNA Polimerase I/genética , DNA Primase/genética , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
4.
Nat Commun ; 12(1): 4826, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376696

RESUMO

Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood-brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.


Assuntos
Barreira Hematoencefálica/metabolismo , Glucose/metabolismo , Complexos Multiproteicos/metabolismo , Quinase 1 Relacionada a NIMA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/metabolismo , Quinase 1 Relacionada a NIMA/genética , Necroptose/genética , Fosforilação , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
5.
Nat Microbiol ; 6(9): 1199-1210, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34413503

RESUMO

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers toxic effectors to kill competitors or subvert some of their key functions. Here, we use transposon directed insertion-site sequencing to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine transfer RNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis and validate the use of a transposon directed insertion site sequencing-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/genética , Complexos Multiproteicos/genética , Ligação Proteica , Sistemas de Secreção Tipo VI/genética
6.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204297

RESUMO

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.


Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Quinases da Família src/química , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/química , Arrestinas/metabolismo , Ativação Enzimática , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
7.
Nat Commun ; 12(1): 4255, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253720

RESUMO

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Complexos Multiproteicos/metabolismo , Animais , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Proliferação de Células , Células HEK293 , Humanos , Meiose , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/metabolismo , Mutação/genética , Fenótipo , Rad51 Recombinase/metabolismo , Troca de Cromátide Irmã , Análise de Sobrevida
8.
J Immunol ; 207(3): 888-901, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290105

RESUMO

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.


Assuntos
Proteínas de Anfíbios/metabolismo , Antivirais/metabolismo , Córnea/patologia , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Fatores Trefoil/metabolismo , Proteínas de Anfíbios/genética , Animais , Anuros , Toxinas Bacterianas/genética , Córnea/virologia , Feminino , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Envelope Viral/metabolismo , Envelope Viral/ultraestrutura , Internalização do Vírus , gama-Cristalinas/química
9.
Nat Microbiol ; 6(9): 1129-1139, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34267357

RESUMO

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Cinética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Oxirredução , Oxirredutases/genética
10.
Nature ; 596(7871): 281-284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290409

RESUMO

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.


Assuntos
Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Leucina/deficiência , Longevidade/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/agonistas , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Methods Mol Biol ; 2276: 227-234, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060045

RESUMO

In mitochondrial oxidative phosphorylation (Ox-Phos), individual electron transport chain complexes are thought to assemble into supramolecular entities termed supercomplexes (SCs). The technique of blue native (BN) gel electrophoresis has emerged as the method of choice for analyzing SCs. However, the process of sample extraction for BN gel analysis is somewhat tedious and introduces the possibility for experimental artifacts. Here we outline a streamlined method that eliminates a centrifugation step and provides a more representative sampling of a population of mitochondria on the final gel. Using this method, we show that SC composition does not appear to change dynamically with altered mitochondrial function.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/análise , Coração/fisiologia , Mitocôndrias Cardíacas/química , Proteínas Mitocondriais/análise , Complexos Multiproteicos/análise , Miocárdio/química , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Animais , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Camundongos , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Miocárdio/metabolismo , Fosforilação Oxidativa
12.
Methods Mol Biol ; 2275: 301-314, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118046

RESUMO

Our group has previously established a strategy utilizing fluorescence lifetime probes to image membrane protein supercomplex (SC) formation in situ. We showed that a probe at the interface between individual mitochondrial respiratory complexes exhibits a decreased fluorescence lifetime when a supercomplex is formed. This is caused by electrostatic interactions with the adjacent proteins. Fluorescence lifetime imaging microscopy (FLIM) records the resulting decrease of the lifetime of the SC-probe. Here we present the details of our method for performing SC-FLIM, including the evaluation of fluorescence lifetimes from the FLIM images. To validate the feasibility of the technique for monitoring adaptive SC formation, we compare data obtained under different metabolic conditions. The results confirm that SC formation is dynamic.


Assuntos
Proteínas de Fluorescência Verde/química , Mitocôndrias/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Moleculares , Conformação Molecular , Multimerização Proteica
13.
Nat Commun ; 12(1): 3748, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145250

RESUMO

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB-CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.


Assuntos
Antígenos/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/patologia , Proteoglicanas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sítios de Ligação/fisiologia , Anticorpos Amplamente Neutralizantes/farmacologia , Microscopia Crioeletrônica , Enterocolite Pseudomembranosa/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteoglicanas/genética
14.
Mol Cell ; 81(15): 3187-3204.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34157307

RESUMO

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.


Assuntos
Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Endopeptidases/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Poliubiquitina/metabolismo , Adulto , Animais , Endopeptidases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Fator 2 de Diferenciação de Crescimento/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Mutantes , Mutação , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Telangiectasia Hemorrágica Hereditária , Ubiquitina-Proteína Ligases/metabolismo
15.
Commun Biol ; 4(1): 667, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083717

RESUMO

Complex formation between hexokinase-II (HKII) and the mitochondrial VDAC1 is crucial to cell growth and survival. We hypothesize that HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we devised a hybrid approach. First, we describe membrane binding of HKII with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion capturing membrane insertion of its H-anchor. The insertion depth of the H-anchor was then used to derive positional restraints in subsequent millisecond-scale Brownian dynamics (BD) simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models for the complex were further refined and their structural stability probed with additional MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1's permeation pathway, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. We also show how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and has implications in mitochondria-mediated cell death.


Assuntos
Hexoquinase/metabolismo , Proteínas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Hexoquinase/química , Hexoquinase/genética , Humanos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Ligação Proteica , Domínios Proteicos , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genética
16.
Viruses ; 13(6)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064113

RESUMO

Flaviviruses are vector-borne RNA viruses, many of which are clinically relevant human viral pathogens, such as dengue, Zika, Japanese encephalitis, West Nile and yellow fever viruses. Millions of people are infected with these viruses around the world each year. Vaccines are only available for some members of this large virus family, and there are no effective antiviral drugs to treat flavivirus infections. The unmet need for vaccines and therapies against these flaviviral infections drives research towards a better understanding of the epidemiology, biology and immunology of flaviviruses. In this review, we discuss the basic biology of the flavivirus replication process and focus on the molecular aspects of viral genome replication. Within the virus-induced intracellular membranous compartments, flaviviral RNA genome replication takes place, starting from viral poly protein expression and processing to the assembly of the virus RNA replication complex, followed by the delivery of the progeny viral RNA to the viral particle assembly sites. We attempt to update the latest understanding of the key molecular events during this process and highlight knowledge gaps for future studies.


Assuntos
Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Flavivirus/fisiologia , Interações Hospedeiro-Patógeno , Complexos Multiproteicos/metabolismo , Replicação Viral , Proteínas de Transporte/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Mapas de Interação de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismo
17.
Nat Commun ; 12(1): 3456, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103492

RESUMO

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.


Assuntos
Proteínas de Transporte/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Dedos de Zinco , Sítios de Ligação , Proteínas de Transporte/química , Sobrevivência Celular , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Schizosaccharomyces/citologia
18.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072647

RESUMO

Steroid-induced glaucoma is a severe pathological condition, sustained by a rapidly progressive increase in intraocular pressure (IOP), which is diagnosed in a subset of subjects who adhere to a glucocorticoid (GC)-based therapy. Molecular and clinical studies suggest that either natural or synthetic GCs induce a severe metabolic dysregulation of Trabecular Meshwork Cells (TMCs), an endothelial-derived histotype with phagocytic and secretive functions which lay at the iridocorneal angle in the anterior segment of the eye. Since TMCs physiologically regulate the composition and architecture of trabecular meshwork (TM), which is the main outflow pathway of aqueous humor, a fluid which shapes the eye globe and nourishes the lining cell types, GCs are supposed to trigger a pathological remodeling of the TM, inducing an IOP increase and retina mechanical compression. The metabolic dysregulation of TMCs induced by GCs exposure has never been characterized at the molecular detail. Herein, we report that, upon dexamethasone exposure, a TMCs strain develops a marked inhibition of the autophagosome biogenesis pathway through an enhanced turnover of two members of the Ulk-1 complex, the main platform for autophagy induction, through the Ubiquitin Proteasome System (UPS).


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Dexametasona/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proliferação de Células/efeitos dos fármacos , Dexametasona/efeitos adversos , Suscetibilidade a Doenças , Glaucoma/etiologia , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexo de Endopeptidases do Proteassoma/metabolismo
19.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063491

RESUMO

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems-cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG, and dual-specificity RegA-RD complexes-and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores de Fosfodiesterase/farmacologia , Extratos Vegetais/farmacologia , Proteínas Quinases/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Domínio Catalítico , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Polarização de Fluorescência , Terapia de Alvo Molecular , Complexos Multiproteicos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato
20.
Nat Commun ; 12(1): 3426, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103516

RESUMO

Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Complexos Multiproteicos/metabolismo , Transcrição Genética , Proteínas de Arabidopsis/genética , Epistasia Genética , Genes de Plantas , Loci Gênicos , Fatores de Transcrição de Choque Térmico/genética , Histonas/metabolismo , Cinética , Lisina/metabolismo , Metilação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...