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1.
Cell ; 186(17): 3632-3641.e10, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37516108

RESUMO

The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.


Assuntos
Proteína ADAM10 , Animais , Proteína ADAM10/química , Proteína ADAM10/metabolismo , Proteína ADAM10/ultraestrutura , Secretases da Proteína Precursora do Amiloide/química , Mamíferos/metabolismo , Proteólise , Tetraspaninas/metabolismo , Humanos
2.
Cell ; 186(26): 5826-5839.e18, 2023 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-38101409

RESUMO

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.


Assuntos
Regulação da Expressão Gênica , Super Intensificadores , Transcrição Gênica , alfa-Globinas , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , alfa-Globinas/genética
3.
Cell ; 186(21): 4546-4566.e27, 2023 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-37769657

RESUMO

Neutrophils are abundant immune cells in the circulation and frequently infiltrate tumors in substantial numbers. However, their precise functions in different cancer types remain incompletely understood, including in the brain microenvironment. We therefore investigated neutrophils in tumor tissue of glioma and brain metastasis patients, with matched peripheral blood, and herein describe the first in-depth analysis of neutrophil phenotypes and functions in these tissues. Orthogonal profiling strategies in humans and mice revealed that brain tumor-associated neutrophils (TANs) differ significantly from blood neutrophils and have a prolonged lifespan and immune-suppressive and pro-angiogenic capacity. TANs exhibit a distinct inflammatory signature, driven by a combination of soluble inflammatory mediators including tumor necrosis factor alpha (TNF-ɑ) and Ceruloplasmin, which is more pronounced in TANs from brain metastasis versus glioma. Myeloid cells, including tumor-associated macrophages, emerge at the core of this network of pro-inflammatory mediators, supporting the concept of a critical myeloid niche regulating overall immune suppression in human brain tumors.

4.
Cell ; 185(12): 2035-2056.e33, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35688132

RESUMO

Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , Doença de Parkinson/metabolismo , Corpos de Processamento , Estabilidade de RNA , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
5.
Cell ; 184(17): 4447-4463.e20, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34363755

RESUMO

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.


Assuntos
Inflamação/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Fator de Necrose Tumoral alfa/farmacologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Autoimunidade/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Morte Celular/efeitos dos fármacos , Citocinas/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Feminino , Células HEK293 , Homozigoto , Humanos , Quinase I-kappa B/metabolismo , Imunofenotipagem , Inflamação/patologia , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Mutação com Perda de Função/genética , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Receptor 3 Toll-Like/metabolismo , Transcriptoma/genética , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia
6.
Cell ; 184(20): 5089-5106.e21, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34555357

RESUMO

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.


Assuntos
Estruturas da Membrana Celular/metabolismo , Microglia/metabolismo , Proteólise , alfa-Sinucleína/metabolismo , Actinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Citoesqueleto/metabolismo , Regulação para Baixo , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/patologia , Microglia/ultraestrutura , Mitocôndrias/metabolismo , Nanotubos , Agregados Proteicos , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/genética
7.
Cell ; 176(4): 805-815.e8, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30639102

RESUMO

Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size.


Assuntos
Divisão Celular/fisiologia , alfa Carioferinas/metabolismo , alfa Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Tamanho Celular , Citoplasma/metabolismo , Lipoilação , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Óvulo/citologia , Fuso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
8.
Immunity ; 57(7): 1696-1709.e10, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38878770

RESUMO

Aicardi-Goutières syndrome (AGS) is an autoinflammatory disease characterized by aberrant interferon (IFN)-α production. The major cause of morbidity in AGS is brain disease, yet the primary source and target of neurotoxic IFN-α remain unclear. Here, we demonstrated that the brain was the primary source of neurotoxic IFN-α in AGS and confirmed the neurotoxicity of intracerebral IFN-α using astrocyte-driven Ifna1 misexpression in mice. Using single-cell RNA sequencing, we demonstrated that intracerebral IFN-α-activated receptor (IFNAR) signaling within cerebral endothelial cells caused a distinctive cerebral small vessel disease similar to that observed in individuals with AGS. Magnetic resonance imaging (MRI) and single-molecule ELISA revealed that central and not peripheral IFN-α was the primary determinant of microvascular disease in humans. Ablation of endothelial Ifnar1 in mice rescued microvascular disease, stopped the development of diffuse brain disease, and prolonged lifespan. These results identify the cerebral microvasculature as a primary mediator of IFN-α neurotoxicity in AGS, representing an accessible target for therapeutic intervention.


Assuntos
Encéfalo , Interferon-alfa , Microvasos , Malformações do Sistema Nervoso , Receptor de Interferon alfa e beta , Animais , Humanos , Camundongos , Interferon-alfa/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/genética , Microvasos/patologia , Malformações do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Células Endoteliais/metabolismo , Camundongos Knockout , Masculino , Feminino , Transdução de Sinais , Camundongos Endogâmicos C57BL , Astrócitos/metabolismo , Modelos Animais de Doenças
9.
Immunity ; 56(9): 2054-2069.e10, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37597518

RESUMO

Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.


Assuntos
Doenças Autoimunes , Ativação Linfocitária , Humanos , Receptor alfa de Ácido Retinoico/genética , Membrana Celular , Receptores de Antígenos de Linfócitos T
10.
Cell ; 169(1): 148-160.e15, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28340340

RESUMO

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Gluconeogênese/efeitos dos fármacos , Hipoglicemiantes/administração & dosagem , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/antagonistas & inibidores , Acetilação , Animais , Glicemia/metabolismo , Células Cultivadas , Glucose/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala , Resistência à Insulina , Camundongos , Fatores de Transcrição de p300-CBP/metabolismo
11.
Cell ; 164(3): 349-52, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26824652

RESUMO

Type I interferon (IFN-I) elicits a complex cascade of events in response to microbial infection. Here, we review recent developments illuminating the large number of IFN-I species and describing their unique biologic functions.


Assuntos
Infecções Bacterianas/imunologia , Interferon Tipo I/metabolismo , Viroses/imunologia , Animais , Infecções Bacterianas/microbiologia , Humanos , Interferon Tipo I/química , Interferon Tipo I/imunologia , Receptor de Interferon alfa e beta/metabolismo , Viroses/virologia
12.
Cell ; 167(4): 1111-1124.e13, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27814508

RESUMO

Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP.


Assuntos
Citocinas/genética , Citocinas/imunologia , Interação Gene-Ambiente , Adolescente , Adulto , Idoso , Envelhecimento , Animais , Artrite/imunologia , Sangue/imunologia , Índice de Massa Corporal , Feminino , Projeto Genoma Humano , Humanos , Infecções/imunologia , Infecções/microbiologia , Infecções/virologia , Inflamação/imunologia , Inflamação/microbiologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Estações do Ano , Caracteres Sexuais
13.
Mol Cell ; 83(18): 3314-3332.e9, 2023 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-37625404

RESUMO

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.


Assuntos
Doenças Neurodegenerativas , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , alfa-Sinucleína/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo
14.
Mol Cell ; 81(21): 4377-4397.e12, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34478647

RESUMO

Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4-3.5 Å) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations ±0-1 and ±3.5-4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes.


Assuntos
Cromossomos/química , Interfase , Metáfase , Nucleossomos/metabolismo , Animais , Comunicação Celular , Ciclo Celular , Divisão Celular , Cromatina/química , Simulação por Computador , Microscopia Crioeletrônica , DNA/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nucleossomos/química , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Xenopus
15.
EMBO J ; 2024 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-39284910

RESUMO

Transcription factors (TFs) regulate gene expression by binding with varying strengths to DNA via their DNA-binding domain. Additionally, some TFs also interact with RNA, which modulates transcription factor binding to chromatin. However, whether RNA-mediated TF binding results in differential transcriptional outcomes remains unknown. In this study, we demonstrate that estrogen receptor α (ERα), a ligand-activated TF, interacts with RNA in a ligand-dependent manner. Defects in RNA binding lead to genome-wide loss of ERα recruitment, particularly at weaker ERα-motifs. Furthermore, ERα mobility in the nucleus increases in the absence of its RNA-binding capacity. Unexpectedly, this increased mobility coincides with robust polymerase loading and transcription of ERα-regulated genes that harbor low-strength motifs. However, highly stable binding of ERα on chromatin negatively impacts ligand-dependent transcription. Collectively, our results suggest that RNA interactions spatially confine ERα on low-affinity sites to fine-tune gene transcription.

16.
Immunity ; 50(3): 600-615.e15, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30824325

RESUMO

CCR7 chemokine receptor stimulation induces rapid but transient dendritic cell (DC) migration toward draining lymph nodes, which is critical for the initiation of protective immunity and maintenance of immune homeostasis. The mechanisms for terminating CCR7-mediated DC migration remain incompletely understood. Here we have identified a long non-coding RNA lnc-Dpf3 whose feedback restrained CCR7-mediated DC migration. CCR7 stimulation upregulated lnc-Dpf3 via removing N6-methyladenosine (m6A) modification to prevent RNA degradation. DC-specific lnc-Dpf3 deficiency increased CCR7-mediated DC migration, leading to exaggerated adaptive immune responses and inflammatory injuries. Mechanistically, CCR7 stimulation activated the HIF-1α transcription factor pathway in DCs, leading to metabolic reprogramming toward glycolysis for DC migration. lnc-Dpf3 directly bound to HIF-1α and suppressed HIF-1α-dependent transcription of the glycolytic gene Ldha, thus inhibiting DC glycolytic metabolism and migratory capacity. We demonstrate a critical role for CCR7-inducible lnc-Dpf3 in coupling epigenetic and metabolic pathways to feedback-control DC migration and inflammatory responses.


Assuntos
Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Glicólise/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Receptores CCR7/genética , Fatores de Transcrição/genética , Imunidade Adaptativa/genética , Animais , Linhagem Celular , Células Dendríticas/patologia , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Inflamação/genética , Inflamação/patologia , Linfonodos/patologia , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Transcrição Gênica/genética , Regulação para Cima/genética
17.
Mol Cell ; 79(1): 68-83.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32533918

RESUMO

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.


Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fos
18.
Trends Biochem Sci ; 48(10): 860-872, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37586999

RESUMO

Telomere maintenance is essential for the genome integrity of eukaryotes, and this function is underpinned by the two-step telomeric DNA synthesis process: telomere G-overhang extension by telomerase and complementary strand fill-in by DNA polymerase alpha-primase (polα-primase). Compared to the telomerase step, the telomere C-strand fill-in mechanism is less understood. Recent studies have provided new insights into how telomeric single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) and polα-primase coordinate to synthesize the telomeric C-strand for telomere overhang fill-in. Cryogenic electron microscopy (cryo-EM) structures of CST-polα-primase complexes have provided additional insights into how they assemble at telomeric templates and de novo synthesize the telomere C-strand. In this review, we discuss how these latest findings coalesce with existing understanding to develop a human telomere C-strand fill-in mechanism model.


Assuntos
DNA Primase , Telomerase , Humanos , Telômero , Complexo Shelterina , Eucariotos
19.
EMBO J ; 42(23): e111122, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37916890

RESUMO

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.


Assuntos
Cálcio , alfa-Sinucleína , Cálcio/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Membrana Celular/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação
20.
Mol Cell ; 73(5): 1001-1014.e8, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30527540

RESUMO

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.


Assuntos
Antiparkinsonianos/farmacologia , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolômica/métodos , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Estearoil-CoA Dessaturase/antagonistas & inibidores , alfa-Sinucleína/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/enzimologia , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Degeneração Neural , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Neurônios/enzimologia , Neurônios/patologia , Ácido Oleico/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ratos Sprague-Dawley , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/metabolismo , alfa-Sinucleína/genética
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