RESUMO
CpG methylation generally occurs on both DNA strands and is essential for mammalian development and differentiation. Until recently, hemimethylation, in which only one strand is methylated, was considered to be simply a transitory state generated during DNA synthesis. The discovery that a subset of CCCTC-binding factor (CTCF) binding sites is heritably hemimethylated suggests that hemimethylation might have an unknown biological function. Here we show that the binding of CTCF is profoundly altered by which DNA strand is methylated and by the specific CTCF binding motif. CpG methylation on the motif strand can inhibit CTCF binding by up to 7-fold, whereas methylation on the opposite strand can stimulate binding by up to 4-fold. Thus, hemimethylation can alter binding by up to 28-fold in a strand-specific manner. The mechanism for sensing methylation on the opposite strand requires two critical residues, V454 and S364, within CTCF zinc fingers 7 and 4. Similar to methylation, CpG hydroxymethylation on the motif strand can inhibit CTCF binding by up to 4-fold. However, hydroxymethylation on the opposite strand removes the stimulatory effect. Strand-specific methylation states may therefore provide a mechanism to explain the transient and dynamic nature of CTCF-mediated chromatin interactions.
Assuntos
Fator de Ligação a CCCTC , Metilação de DNA , Proteínas Repressoras , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Cromatina , Ilhas de CpG , DNA/metabolismo , Mamíferos/genética , Proteínas Repressoras/metabolismoRESUMO
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas , Modelos Moleculares , Fosforilação , Conformação Proteica , Domínios Proteicos , Engenharia de ProteínasRESUMO
CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.
Assuntos
Microscopia Crioeletrônica , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Nucleossomos/metabolismo , Animais , Biocatálise , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Xenopus/genética , DNA Metiltransferase 3BRESUMO
Formylpeptide receptors (FPRs) as G protein-coupled receptors (GPCRs) can recognize formylpeptides derived from pathogens or host cells to function in host defense and cell clearance. In addition, FPRs, especially FPR2, can also recognize other ligands with a large chemical diversity generated at different stages of inflammation to either promote or resolve inflammation in order to maintain a balanced inflammatory response. The mechanism underlying promiscuous ligand recognition and activation of FPRs is not clear. Here we report a cryo-EM structure of FPR2-Gi signaling complex with a peptide agonist. The structure reveals a widely open extracellular region with an amphiphilic environment for ligand binding. Together with computational docking and simulation, the structure suggests a molecular basis for the recognition of formylpeptides and a potential mechanism of receptor activation, and reveals conserved and divergent features in Gi coupling. Our results provide a basis for understanding the molecular mechanism of the functional promiscuity of FPRs.
Assuntos
Receptores de Formil Peptídeo/química , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/química , Receptores de Lipoxinas/metabolismo , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutação , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Ratos , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/genética , Transdução de SinaisRESUMO
Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Receptor CB2 de Canabinoide/química , Transdução de Sinais , Animais , Sítios de Ligação , Células CHO , Agonistas de Receptores de Canabinoides/síntese química , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/síntese química , Antagonistas de Receptores de Canabinoides/farmacologia , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/metabolismo , Células Sf9 , SpodopteraRESUMO
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.
Assuntos
Receptores Frizzled/química , Sítios de Ligação , Cristalografia por Raios X , Cisteína/metabolismo , Proteínas Desgrenhadas/metabolismo , Receptores Frizzled/genética , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Via de Sinalização WntRESUMO
One of the hallmarks of Alzheimer's disease is the formation of extracellular amyloid plaques that consist mainly of abnormally aggregated forms of amyloid ß (Aß) peptides. These peptides are generated by γ-secretase-catalyzed cleavage of a dimeric membrane-bound C-terminal fragment (C99) of the amyloid precursor protein. Although C99 homodimerization has been linked to Aß production and changes in the aggregation-determining Aß42/Aß40 ratio, the motif through which C99 dimerizes has remained controversial. Here, we have used two independent assays to gain insight into C99 homodimerization in the context of the membrane of live cells: bioluminescence resonance energy transfer and Tango membrane protein-protein interaction assays, which were further confirmed by traditional pull-down assays. Our results indicate a four-amino acid region within the C99 transmembrane helix (43TVIV46) as well as its local secondary structure as critical determinants for homodimerization. These four amino acids are also a hot spot of familial Alzheimer's disease-linked mutations that both decrease C99 homodimerization and γ-secretase cleavage and alter the initial cleavage site to increase the Aß42/40 ratio.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteólise , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Sítios de Ligação , Linhagem Celular , Humanos , Mutagênese , Fragmentos de Peptídeos/genética , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Amyloid beta peptide (Aß) is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by ß- and γ-secretases. Aß accumulation in the brain is proposed to be an early toxic event in the pathogenesis of Alzheimer's disease, which is the most common form of dementia associated with plaques and tangles in the brain. Currently, it is unclear what the physiological and pathological forms of Aß are and by what mechanism Aß causes dementia. Moreover, there are no efficient drugs to stop or reverse the progression of Alzheimer's disease. In this paper, we review the structures, biological functions, and neurotoxicity role of Aß. We also discuss the potential receptors that interact with Aß and mediate Aß intake, clearance, and metabolism. Additionally, we summarize the therapeutic developments and recent advances of different strategies for treating Alzheimer's disease. Finally, we will report on the progress in searching for novel, potentially effective agents as well as selected promising strategies for the treatment of Alzheimer's disease. These prospects include agents acting on Aß, its receptors and tau protein, such as small molecules, vaccines and antibodies against Aß; inhibitors or modulators of ß- and γ-secretase; Aß-degrading proteases; tau protein inhibitors and vaccines; amyloid dyes and microRNAs.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Anticorpos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Vacinas/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos/química , Humanos , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Vacinas/químicaRESUMO
TOPLESS are tetrameric plant corepressors of the conserved Tup1/Groucho/TLE (transducin-like enhancer of split) family. We show that they interact through their TOPLESS domains (TPDs) with two functionally important ethylene response factor-associated amphiphilic repression (EAR) motifs of the rice strigolactone signaling repressor D53: the universally conserved EAR-3 and the monocot-specific EAR-2. We present the crystal structure of the monocot-specific EAR-2 peptide in complex with the TOPLESS-related protein 2 (TPR2) TPD, in which the EAR-2 motif binds the same TPD groove as jasmonate and auxin signaling repressors but makes additional contacts with a second TPD site to mediate TPD tetramer-tetramer interaction. We validated the functional relevance of the two TPD binding sites in reporter gene assays and in transgenic rice and demonstrate that EAR-2 binding induces TPD oligomerization. Moreover, we demonstrate that the TPD directly binds nucleosomes and the tails of histones H3 and H4. Higher-order assembly of TPD complexes induced by EAR-2 binding markedly stabilizes the nucleosome-TPD interaction. These results establish a new TPD-repressor binding mode that promotes TPD oligomerization and TPD-nucleosome interaction, thus illustrating the initial assembly of a repressor-corepressor-nucleosome complex.
Assuntos
Motivos de Aminoácidos , Proteínas Correpressoras/química , Proteínas Correpressoras/metabolismo , Nucleossomos/metabolismo , Multimerização Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Histonas/química , Histonas/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
γ-Secretase is an intramembrane aspartyl protease that cleaves the C99 fragment of amyloid precursor protein to generate extracellular Aß peptides. These peptides can oligomerize and aggregate to form amyloid plaques, processes that are widely believed to be causal for Alzheimer's disease. In spite of this critical function, it remains unknown how γ-secretase recognizes C99 and its other substrates, including Notch. In this study we determined E22-K55 as the minimal C99 fragment that was sufficient and required for initial cleavage. Within this fragment, we identified four determinants: (i) a transferable extracellular determinant that differed between C99 and Notch, and which included negative charge in the case of C99, (ii) the amino acid sequence of the C-terminal half of the transmembrane helix, (iii) an invariant lysine or arginine at the intracellular membrane border, and (iv) a positive charge cluster that included the invariant lysine/arginine. We demonstrated that the charge clusters of C99 and Notch receptors could directly bind phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-binding cluster was required for γ-secretase cleavage, and modulation of membrane PIP2 levels strongly affected γ-secretase cleavage levels and the Aß40/Aß42 ratio, providing support for the importance of the PIP2 interaction in cells. Together, these studies provide critically needed insight into substrate recognition by γ-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Notch/metabolismo , Doença de Alzheimer/fisiopatologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Células Cultivadas , Humanos , Especificidade por SubstratoRESUMO
γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO operator element, in which C99 C terminally fused to a reversed tetracyclin-inducible activator (rTA) transcriptional activator is expressed. Endogenous or transfected γ-secretase cleaves a C terminally fused rTA transcriptional activator from C99, allowing rTA to move to the nucleus to activate a luciferase reporter gene as a measurement for γ-secretase cleavage activity.
RESUMO
The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells.
RESUMO
Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and co-associated Flag-tagged protein using Streptavidin MagBeads.
RESUMO
The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins tagged with bioluminescent Renilla luciferase (Rlu) as donor and yellow fluorescent protein (YFP) as acceptor were co-expressed in cells. This pair of donor and acceptor have an approximate Förster distance of 4.4 nm, providing the optimal working distance (Dacres et al., 2010). This technique can be used to explore the time-course of specific molecular interactions that occur in living cells.
RESUMO
Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by γ-secretase are associated with Alzheimer's disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by γ-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to γ-secretase. Mutations that alter residues N terminal to the γ-secretase cleavage site Aß42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the Aß42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone Aß42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by γ-secretase. These results suggest a model of γ-secretase substrate recognition, in which the APP region C terminal to the Aß42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to γ-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins.