Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 14.716
Filtrar
3.
Nat Struct Mol Biol ; 28(10): 825-834, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34625749

RESUMO

Xkr8-Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8-Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8-Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.


Assuntos
Proteínas Reguladoras de Apoptose/química , Basigina/química , Membrana Celular/metabolismo , Proteínas de Membrana/química , Fosfolipídeos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Basigina/metabolismo , Microscopia Crioeletrônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/química , Estrutura Terciária de Proteína , Triptofano/química
4.
Nat Struct Mol Biol ; 28(10): 847-857, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34625747

RESUMO

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.


Assuntos
Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Galectinas/química , Galectinas/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutagênese , Multimerização Proteica , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
5.
Nat Commun ; 12(1): 5865, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620869

RESUMO

Condensation of hundreds of mega-base-pair-long human chromosomes in a small nuclear volume is a spectacular biological phenomenon. This process is driven by the formation of chromosome loops. The ATP consuming motor, condensin, interacts with chromatin segments to actively extrude loops. Motivated by real-time imaging of loop extrusion (LE), we created an analytically solvable model, predicting the LE velocity and step size distribution as a function of external load. The theory fits the available experimental data quantitatively, and suggests that condensin must undergo a large conformational change, induced by ATP binding, bringing distant parts of the motor to proximity. Simulations using a simple model confirm that the motor transitions between an open and a closed state in order to extrude loops by a scrunching mechanism, similar to that proposed in DNA bubble formation during bacterial transcription. Changes in the orientation of the motor domains are transmitted over ~50 nm, connecting the motor head and the hinge, thus providing an allosteric basis for LE.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Complexos Multiproteicos/metabolismo , Bactérias/genética , Cromatina , Cromossomos/metabolismo , DNA/química , Técnicas Genéticas , Humanos , Cinética , Modelos Genéticos , Transcrição Genética
6.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639046

RESUMO

Here, we review recent molecular modelling and simulation studies of the Sec translocon, the primary component/channel of protein translocation into the endoplasmic reticulum (ER) and bacterial periplasm, respectively. Our focus is placed on the eukaryotic Sec61, but we also mention modelling studies on prokaryotic SecY since both systems operate in related ways. Cryo-EM structures are now available for different conformational states of the Sec61 complex, ranging from the idle or closed state over an inhibited state with the inhibitor mycolactone bound near the lateral gate, up to a translocating state with bound substrate peptide in the translocation pore. For all these states, computational studies have addressed the conformational dynamics of the translocon with respect to the pore ring, the plug region, and the lateral gate. Also, molecular simulations are addressing mechanistic issues of insertion into the ER membrane vs. translocation into the ER, how signal-peptides are recognised at all in the translocation pore, and how accessory proteins affect the Sec61 conformation in the co- and post-translational pathways.


Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Peptídeos/química , Sinais Direcionadores de Proteínas , Canais de Translocação SEC/química , Sequência de Aminoácidos , Animais , Eucariotos , Células Eucarióticas/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Canais de Translocação SEC/metabolismo , Relação Estrutura-Atividade
7.
Nutrients ; 13(10)2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34684518

RESUMO

BACKGROUND: Children and adolescents affected by type 1 diabetes have an increased risk of being overweight or obese and of suffering from cardiometabolic symptoms. AIMS: To retrospectively evaluate the effects of a new complex of polysaccharide macromolecules, Policaptil Gel Retard® (PGR), on auxological and metabolic parameters, glycaemic variability and control parameters in paediatric patients with type 1 diabetes and metabolic syndrome (MetS). PATIENTS AND METHODS: Data for 27 paediatric patients with a diagnosis of type 1 diabetes in conjunction with obesity and MetS of at least 5 years' standing were collected and retrospectively studied. Of these, 16 (median age 12.9, range 9.5-15.8 years) had been adjunctively treated with PGR and 11 (median age 12.6, range 9.4-15.6 years) had not been treated with PGR. Auxological, metabolic and glycaemic control and variability parameters and insulin dosing were compared after 6 months in the two groups. RESULTS: PGR significantly reduced BMI standard deviation score (SDS) (p < 0.005), waist SDS (p < 0.005), HbA1c (p < 0.05) and daily mean insulin dose requirement (p < 0.005). A significant improvement was also observed in the metabolic and glycaemic variability parameters of mean daily blood glucose (BG) levels (p < 0.005), SD of daily BG levels (p < 0.0001), mean coefficient of variation (p < 0.05), LBGI (p < 0.0001), HBGI (p < 0.0001), J-index (p < 0.005), total cholesterol (p < 0.005), HDL-cholesterol (p < 0.005) and LDL-cholesterol (p < 0.005) and triglycerides (p < 0.05). CONCLUSIONS: PGR produces a good auxological and metabolic response in obese patients with MetS who are affected by type 1 diabetes. It led to a significant reduction in BMI SDS, waist SDS and an improvement in glucose control and variability as well as in other MetS parameters. The use of polysaccharide compounds, especially if associated with appropriate dietary changes, may help achieve treatment targets in type 1 diabetes and reduce the risk that patients develop metabolic syndrome.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Síndrome Metabólica/tratamento farmacológico , Obesidade Pediátrica/tratamento farmacológico , Polissacarídeos/administração & dosagem , Adolescente , Glicemia/metabolismo , Índice de Massa Corporal , Criança , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Feminino , Humanos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/complicações , Complexos Multiproteicos , Obesidade Pediátrica/sangue , Obesidade Pediátrica/complicações , Estudos Retrospectivos , Resultado do Tratamento , Triglicerídeos/sangue
8.
Int J Mol Sci ; 22(19)2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34639003

RESUMO

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein-protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes' specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.


Assuntos
Proteínas Sanguíneas/metabolismo , Butirilcolinesterase/metabolismo , Proteínas Sanguíneas/química , Butirilcolinesterase/química , Proteínas de Transporte , Centrifugação com Gradiente de Concentração/métodos , HDL-Colesterol , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Ativação Enzimática , Humanos , Imunoprecipitação , Espectrometria de Massas , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Ligação Proteica , Especificidade por Substrato
9.
Nat Commun ; 12(1): 5767, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599184

RESUMO

Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disability in young females. RTT is mainly caused by mutations found in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Despite extensive studies, the molecular mechanism underlying RTT pathogenesis is still poorly understood. Here, we report MeCP2 as a key subunit of a higher-order multiunit protein complex Rbfox/LASR. Defective MeCP2 in RTT mouse models disrupts the assembly of the MeCP2/Rbfox/LASR complex, leading to reduced binding of Rbfox proteins to target pre-mRNAs and aberrant splicing of Nrxns and Nlgn1 critical for synaptic plasticity. We further show that MeCP2 disease mutants display defective condensate properties and fail to promote phase-separated condensates with Rbfox proteins in vitro and in cultured cells. These data link an impaired function of MeCP2 with disease mutation in splicing control to its defective properties in mediating the higher-order assembly of the MeCP2/Rbfox/LASR complex.


Assuntos
Proteína 2 de Ligação a Metil-CpG/metabolismo , Complexos Multiproteicos/metabolismo , Fatores de Processamento de RNA/metabolismo , Síndrome de Rett/genética , Processamento Alternativo/genética , Animais , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Éxons/genética , Feminino , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/química , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Domínios Proteicos , Subunidades Proteicas/metabolismo
10.
Elife ; 102021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34636324

RESUMO

Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1ß- or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1ß or TLR stimulus dynamically induced the formation of Regnase-1-ß-transducin repeat-containing protein (ßTRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. In IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 is known to be required for the recognition by ßTRCP and proteasome-mediated degradation. We found that Regnase-1-14-3-3 and Regnase-1-ßTRCP interactions are not sequential events. Rather, 14-3-3 protects Regnase-1 from ßTRCP-mediated degradation. On the other hand, 14-3-3 abolishes Regnase-1-mediated mRNA decay by inhibiting Regnase-1-mRNA association. In addition, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/genética , Complexos Multiproteicos/genética , Estabilidade de RNA/genética , RNA Mensageiro/química , Ribonucleases/genética , Animais , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos , Complexos Multiproteicos/metabolismo , Ribonucleases/metabolismo
11.
Nature ; 598(7880): 368-372, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34526721

RESUMO

Transcription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription-repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.


Assuntos
Microscopia Crioeletrônica , Reparo do DNA , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Transcrição Genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação
12.
J Biol Chem ; 297(4): 101148, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34473993

RESUMO

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund-Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , RecQ Helicases/metabolismo , Hidrolases Anidrido Ácido/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteína Homóloga a MRE11/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , RecQ Helicases/genética
13.
Plant Mol Biol ; 107(3): 147-158, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34562198

RESUMO

KEY MESSAGE: The GAF1 transcription factor is shown to bind to the promoter of the Arabidopsis GA-biosynthetic enzyme GA20ox1 and, in association with DELLA protein, promotes GA20ox1 expression, thereby contributing to its feedback regulation and tissue specificity. Gibberellins (GAs) are phytohormones that promote plant growth and development, including germination, elongation, flowering, and floral development. Homeostasis of endogenous GA levels is controlled by GA feedback regulation. DELLAs are negative regulators of GA signaling that are rapidly degraded in the presence of GAs. DELLAs regulate several target genes, including AtGA20ox2 and AtGA3ox1, encoding the GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, respectively. Previous studies have identified GAI-ASSOCIATED FACTOR 1 (GAF1) as a DELLA interactor, with which DELLAs act as transcriptional coactivators; furthermore, AtGA20ox2, AtGA3ox1, and AtGID1b were identified as target genes of the DELLA-GAF1 complex. Among the five Arabidopsis GA20ox genes, AtGA20ox1 is the most highly expressed gene during vegetative growth; its expression is controlled by GA feedback regulation. Here, we investigated whether AtGA20ox1 is regulated by the DELLA-GAF1 complex. The electrophoretic mobility shift and transactivation assays showed that three GAF1-binding sites exist in the AtGA20ox1 promoter. Using transgenic plants, we further evaluated the contribution of the DELLA-GAF1 complex to GA feedback regulation and tissue-specific expression. Mutations in two GAF1-binding sites obliterated the negative feedback regulation and tissue-specific expression of AtGA20ox1 in transgenic plants. Thus, our results showed that GAF1-binding sites are involved in GA feedback regulation and tissue-specific expression of AtGA20ox1 in Arabidopsis, suggesting that the DELLA-GAF1 complex is involved in both processes.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Giberelinas/metabolismo , Oxigenases de Função Mista/genética , Arabidopsis/efeitos dos fármacos , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Retroalimentação Fisiológica , Flores/genética , Regulação da Expressão Gênica de Plantas , Giberelinas/farmacologia , Oxigenases de Função Mista/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas
14.
Int J Mol Sci ; 22(17)2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34502494

RESUMO

The study of protein-protein interactions is of great interest. Several early studies focused on the murine double minute 2 (Mdm2)-tumor suppressor protein p53 interactions. However, the effect of plasma treatment on Mdm2 and p53 is still absent from the literature. This study investigated the structural changes in Mdm2, p53, and the Mdm2-p53 complex before and after possible plasma oxidation through molecular dynamic (MD) simulations. MD calculation revealed that the oxidized Mdm2 bounded or unbounded showed high flexibility that might increase the availability of tumor suppressor protein p53 in plasma-treated cells. This study provides insight into Mdm2 and p53 for a better understanding of plasma oncology.


Assuntos
Complexos Multiproteicos/química , Gases em Plasma/química , Proteínas Proto-Oncogênicas c-mdm2/química , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Oxigênio/química , Proteína Supressora de Tumor p53/química , Humanos
15.
Elife ; 102021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34468314

RESUMO

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.


Assuntos
Bactérias/ultraestrutura , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Membrana Externa Bacteriana/ultraestrutura , Extensões da Superfície Celular/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Bactérias/classificação , Complexos Multiproteicos
16.
Nat Commun ; 12(1): 5595, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552083

RESUMO

By incorporating two mutually exclusive factors, PID-1 and TOST-1, C. elegans PICS complex plays important roles in piRNA biogenesis, chromosome segregation and cell division. We firstly map the interaction network between PICS subunits, then uncover the mechanisms underlying the interactions between PICS subunits by solving several complex structures, including those of TOFU-6/PICS-1, ERH-2/PICS-1, and ERH-2/TOST-1. Our biochemical experiment also demonstrates that PICS exists as an octamer consisting of two copies of each subunit. Combining structural analyses with mutagenesis experiments, we identify interfacial residues of PICS subunits that are critical for maintaining intact PICS complex in vitro. Furthermore, using genetics, cell biology and imaging experiments, we find that those mutants impairing the in vitro interaction network within PICS, also lead to dysfunction of PICS in vivo, including mislocalization of PICS, and reduced levels of piRNAs or aberrant chromosome segregation and cell division. Therefore, our work provides structural insights into understanding the PICS-mediated piRNA biogenesis and cell division.


Assuntos
Proteínas de Caenorhabditis elegans/química , Divisão Celular , RNA Interferente Pequeno/biossíntese , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Cristalografia por Raios X , Espaço Intracelular/metabolismo , Complexos Multiproteicos , Mutagênese , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas
17.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34576029

RESUMO

Natural genetic variation in photosynthesis is strictly associated with the remarkable adaptive plasticity observed amongst Arabidopsis thaliana accessions derived from environmentally distinct regions. Exploration of the characteristic features of the photosynthetic machinery could reveal the regulatory mechanisms underlying those traits. In this study, we performed a detailed characterisation and comparison of photosynthesis performance and spectral properties of the photosynthetic apparatus in the following selected Arabidopsis thaliana accessions commonly used in laboratories as background lines: Col-0, Col-1, Col-2, Col-8, Ler-0, and Ws-2. The main focus was to distinguish the characteristic disparities for every accession in photosynthetic efficiency that could be accountable for their remarkable plasticity to adapt. The biophysical and biochemical analysis of the thylakoid membranes in control conditions revealed differences in lipid-to-protein contribution, Chlorophyll-to-Carotenoid ratio (Chl/Car), and xanthophyll cycle pigment distribution among accessions. We presented that such changes led to disparities in the arrangement of the Chlorophyll-Protein complexes, the PSI/PSII ratio, and the lateral mobility of the thylakoid membrane, with the most significant aberrations detected in the Ler-0 and Ws-2 accessions. We concluded that selecting an accession suitable for specific research on the photosynthetic process is essential for optimising the experiment.


Assuntos
Arabidopsis/genética , Carotenoides , Clorofila/genética , Fotossíntese/genética , Seleção Genética/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/ultraestrutura , Regulação da Expressão Gênica de Plantas , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Mutação/genética , Fenótipo , Tilacoides/genética , Tilacoides/ultraestrutura
18.
J Biol Chem ; 297(4): 101123, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34492270

RESUMO

Histone arginine methylation is a key post-translational modification that mediates epigenetic events that activate or repress gene transcription. Protein arginine methyltransferases (PRMTs) are the driving force for the process of arginine methylation, and the core histone proteins have been shown to be substrates for most PRMT family members. However, previous reports of the enzymatic activities of PRMTs on histones in the context of nucleosomes seem contradictory. Moreover, what governs nucleosomal substrate recognition of different PRMT members is not understood. We sought to address this key biological question by examining how different macromolecular contexts where the core histones reside may regulate arginine methylation catalyzed by individual PRMT members (i.e., PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, and PRMT8). Our results demonstrated that the substrate context exhibits a huge impact on the histone arginine methylation activity of PRMTs. Although all the tested PRMTs methylate multiple free histones individually, they show a preference for one particular histone substrate in the context of the histone octamer. We found that PRMT1, PRMT3, PRMT5, PRMT6, PRMT7, and PRMT8 preferentially methylate histone H4, whereas PRMT4/coactivator-associated arginine methyltransferase 1 prefers histone H3. Importantly, neither reconstituted nor cell-extracted mononucleosomes could be methylated by any PRMTs tested. Structural analysis suggested that the electrostatic interaction may play a mechanistic role in priming the substrates for methylation by PRMT enzymes. Taken together, this work expands our knowledge on the molecular mechanisms of PRMT substrate recognition and has important implications for understanding cellular dynamics and kinetics of histone arginine methylation in regulating gene transcription and other chromatin-templated processes.


Assuntos
Histonas/química , Complexos Multiproteicos/química , Proteína-Arginina N-Metiltransferases/química , Arginina/química , Arginina/genética , Arginina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Estrutura Quaternária de Proteína , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Especificidade por Substrato
19.
Nat Struct Mol Biol ; 28(8): 671-680, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373645

RESUMO

Meiotic cells invoke breast cancer susceptibility gene 2 (BRCA2) to repair programmed double-stranded DNA breaks and accomplish homologous recombination. The meiosis-specific protein MEILB2 facilitates BRCA2 recruitment to meiotic recombination sites. Here, we combine crystallography, biochemical analysis and a mouse meiosis model to reveal a robust architecture that ensures meiotic BRCA2 recruitment. The crystal structure of the MEILB2-BRCA2 complex reveals how two MEILB2 homodimers sandwich two chains of BRCA2 to afford a 4:2 architecture. The sandwich lacks close contact between the two MEILB2 dimers or the two BRCA2 chains. Instead, the two halves of each BRCA2 chain bridge two MEILB2 subunits from different homodimers to form the MEILB2-BRCA2-MEILB2 sandwich. Several identical residues from the two MEILB2 subunits are employed to engage the BRCA2 halves, justifying their strict conservation. Mutational analysis of the interface reveals a synergistic mechanism for MEILB2-BRCA2 recruitment during meiosis. Overall, these studies demonstrate how BRCA2 efficiently localizes in the cell to facilitate meiosis.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Meiose/genética , Reparo de DNA por Recombinação/genética , Animais , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...