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1.
Mol Cell ; 82(3): 598-615.e8, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34998453

RESUMO

An increasing number of genetic diseases are linked to deregulation of E3 ubiquitin ligases. Loss-of-function mutations in the RING-between-RING (RBR) family E3 ligase RNF216 (TRIAD3) cause Gordon-Holmes syndrome (GHS) and related neurodegenerative diseases. Functionally, RNF216 assembles K63-linked ubiquitin chains and has been implicated in regulation of innate immunity signaling pathways and synaptic plasticity. Here, we report crystal structures of key RNF216 reaction states including RNF216 in complex with ubiquitin and its reaction product, K63 di-ubiquitin. Our data provide a molecular explanation for chain-type specificity and reveal the molecular basis for disruption of RNF216 function by pathogenic GHS mutations. Furthermore, we demonstrate how RNF216 activity and chain-type specificity are regulated by phosphorylation and that RNF216 is allosterically activated by K63-linked di-ubiquitin. These molecular insights expand our understanding of RNF216 function and its role in disease and further define the mechanistic diversity of the RBR E3 ligase family.


Assuntos
Ataxia Cerebelar/enzimologia , Hormônio Liberador de Gonadotropina/deficiência , Hipogonadismo/enzimologia , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Regulação Alostérica , Sítios de Ligação , Catálise , Ataxia Cerebelar/genética , Cristalografia por Raios X , Predisposição Genética para Doença , Hormônio Liberador de Gonadotropina/genética , Células HEK293 , Humanos , Hipogonadismo/genética , Mutação com Perda de Função , Lisina , Modelos Moleculares , Fenótipo , Fosforilação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
2.
Genes Dev ; 32(21-22): 1420-1429, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30366906

RESUMO

Mutations in Trp53, prevalent in human cancer, are reported to drive tumorigenesis through dominant-negative effects (DNEs) over wild-type TRP53 function as well as neomorphic gain-of-function (GOF) activity. We show that five TRP53 mutants do not accelerate lymphomagenesis on a TRP53-deficient background but strongly synergize with c-MYC overexpression in a manner that distinguishes the hot spot Trp53 mutations. RNA sequencing revealed that the mutant TRP53 DNE does not globally repress wild-type TRP53 function but disproportionately impacts a subset of wild-type TRP53 target genes. Accordingly, TRP53 mutant proteins impair pathways for DNA repair, proliferation, and metabolism in premalignant cells. This reveals that, in our studies of lymphomagenesis, mutant TRP53 drives tumorigenesis primarily through the DNE, which modulates wild-type TRP53 function in a manner advantageous for neoplastic transformation.


Assuntos
Carcinogênese/genética , Mutação , Proteína Supressora de Tumor p53/genética , Animais , Linfoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Supressora de Tumor p53/metabolismo
3.
EMBO J ; 40(20): e107237, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34523147

RESUMO

BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting.


Assuntos
Lipossomos/química , Lipídeos de Membrana/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/química , Animais , Sítios de Ligação , Clonagem Molecular , Medição da Troca de Deutério , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , Camundongos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
4.
Mol Cell ; 68(4): 659-672.e9, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29149594

RESUMO

Certain BH3-only proteins transiently bind and activate Bak and Bax, initiating their oligomerization and the permeabilization of the mitochondrial outer membrane, a pivotal step in the mitochondrial pathway to apoptosis. Here we describe the first crystal structures of an activator BH3 peptide bound to Bak and illustrate their use in the design of BH3 derivatives capable of inhibiting human Bak on mitochondria. These BH3 derivatives compete for the activation site at the canonical groove, are the first engineered inhibitors of Bak activation, and support the role of key conformational transitions associated with Bak activation.


Assuntos
Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2 , Mitocôndrias , Peptídeos , Proteína Killer-Antagonista Homóloga a bcl-2 , Animais , Proteína 11 Semelhante a Bcl-2/química , Proteína 11 Semelhante a Bcl-2/farmacologia , Linhagem Celular Transformada , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
5.
EMBO J ; 39(18): e106275, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32845033

RESUMO

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.


Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , Ubiquitina/metabolismo , Animais , Sítios de Ligação , Chlorocebus aethiops , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Cristalografia por Raios X , Citocinas/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos , Polarização de Fluorescência , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Inibidores de Proteases/farmacologia , Conformação Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Ubiquitinas/genética , Células Vero
6.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30573668

RESUMO

The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co-ordinate the removal of damaged mitochondria. We report that following mitochondrial damage-induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3-only proteins and its homo-dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX-mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK-dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1-dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro-inflammatory effect.


Assuntos
Mitocôndrias/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Camundongos , Mitofagia , Ubiquitinação , Proteína Killer-Antagonista Homóloga a bcl-2/química
7.
Biochem Soc Trans ; 49(1): 365-377, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33634825

RESUMO

The accumulation of misfolded proteins is associated with numerous degenerative conditions, cancers and genetic diseases. These pathological imbalances in protein homeostasis (termed proteostasis), result from the improper triage and disposal of damaged and defective proteins from the cell. The ubiquitin-proteasome system is a key pathway for the molecular control of misfolded cytosolic proteins, co-opting a cascade of ubiquitin ligases to direct terminally damaged proteins to the proteasome via modification with chains of the small protein, ubiquitin. Despite the evidence for ubiquitination in this critical pathway, the precise complement of ubiquitin ligases and deubiquitinases that modulate this process remains under investigation. Whilst chaperones act as the first line of defence against protein misfolding, the ubiquitination machinery has a pivotal role in targeting terminally defunct cytosolic proteins for destruction. Recent work points to a complex assemblage of chaperones, ubiquitination machinery and subcellular quarantine as components of the cellular arsenal against proteinopathies. In this review, we examine the contribution of these pathways and cellular compartments to the maintenance of the cytosolic proteome. Here we will particularly focus on the ubiquitin code and the critical enzymes which regulate misfolded proteins in the cytosol, the molecular point of origin for many neurodegenerative and genetic diseases.


Assuntos
Citosol/metabolismo , Proteostase/fisiologia , Ubiquitinação/fisiologia , Animais , Citoplasma/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Proteólise , Ubiquitina/metabolismo
8.
J Biol Chem ; 294(45): 16525-16526, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704773

RESUMO

Cellular processes accompanying protein aggregation are diverse and entangled, making it difficult to investigate the underlying molecular processes in a time-resolved way. Gottlieb, Thompson, and colleagues address this shortcoming using a chemical biology approach to monitor ubiquitination within the first 10 min after the initiation of protein aggregation. Intriguingly, unfolding rather than aggregation seems to trigger the observed events. This work might provide a method to answer open questions regarding the regulation of the proteostasis network upon protein misfolding.


Assuntos
Agregados Proteicos , Proteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Desdobramento de Proteína , Proteínas/química , Proteostase , Ubiquitinação
9.
Blood ; 132(2): 197-209, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29784641

RESUMO

The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.


Assuntos
Apoptose , Plaquetas/metabolismo , Suscetibilidade a Doenças , Animais , Apoptose/genética , Biomarcadores , Tempo de Sangramento , Contagem de Células Sanguíneas , Coagulação Sanguínea , Caspases/metabolismo , Sobrevivência Celular/genética , Feminino , Genótipo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
10.
J Mol Biol ; 432(7): 2349-2368, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32105731

RESUMO

Recently generated proteomic data provides unprecedented insight into stress granule composition and stands as fruitful ground for further analysis. Stress granules are stress-induced biological assemblies that are of keen interest due to being linked to both long-term cell viability and a variety of protein aggregation-based diseases. Herein, we compile recently published stress granule composition data, formed specifically through heat and oxidative stress, for both mammalian (Homo sapiens) and yeast (Saccharomyces cerevisiae) cells. Interrogation of the data reveals that stress granule proteins are enriched in features that favor protein liquid-liquid phase separation, being highly disordered, soluble, and abundant while maintaining a high level of protein-protein interactions under basal conditions. Furthermore, these "stress granuleomes" are shown to be enriched for multidomained, RNA-binding proteins with increased potential for post-translational modifications. Findings are consistent with the notion that stress granule formation is driven by protein liquid-liquid phase separation. Furthermore, stress granule proteins appear poised near solubility limits while possessing the ability to dynamically alter their phase behavior in response to external threat. Interestingly, several features, such as protein disorder, are more prominent among stress granule proteins that share homologs between yeast and mammalian systems also found within stress-induced foci. We culminate results from our stress granule analysis into novel predictors for granule incorporation and validate the mammalian predictor's performance against multiple types of membraneless condensates and by colocalization microscopy.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Choque Térmico/metabolismo , Organelas/metabolismo , Proteoma/análise , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Células HeLa , Humanos
11.
Nat Struct Mol Biol ; 27(11): 1024-1031, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32929280

RESUMO

BAK and BAX are essential mediators of apoptosis that oligomerize in response to death cues, thereby causing permeabilization of the mitochondrial outer membrane. Their transition from quiescent monomers to pore-forming oligomers involves a well-characterized symmetric dimer intermediate. However, no essential secondary interface that can be disrupted by mutagenesis has been identified. Here we describe crystal structures of human BAK core domain (α2-α5) dimers that reveal preferred binding sites for membrane lipids and detergents. The phospholipid headgroup and one acyl chain (sn2) associate with one core dimer while the other acyl chain (sn1) associates with a neighboring core dimer, suggesting a mechanism by which lipids contribute to the oligomerization of BAK. Our data support a model in which, unlike for other pore-forming proteins whose monomers assemble into oligomers primarily through protein-protein interfaces, the membrane itself plays a role in BAK and BAX oligomerization.


Assuntos
Lipídeos de Membrana/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Lipídeos de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química
12.
Cell Rep ; 22(6): 1496-1508, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29425505

RESUMO

Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling in vivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways.


Assuntos
Infecções Bacterianas/imunologia , Imunidade Inata/imunologia , Proteínas Inibidoras de Apoptose/imunologia , Transdução de Sinais/imunologia , Animais , Proteínas de Bactérias/imunologia , Técnicas de Inativação de Genes , Camundongos , Proteína Adaptadora de Sinalização NOD2 , Peptidoglicano/imunologia
13.
Cell Death Dis ; 8(7): e2914, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28682312

RESUMO

Anaemia is a major global health problem arising from diverse causes and for which improved therapeutic strategies are needed. Erythroid cells can undergo apoptotic cell death and loss of pro-survival BCL-XL is known to trigger apoptosis during late-stage erythroid development. However, the mechanism by which loss or pharmacological blockade of BCL-XL leads to erythroid cell apoptosis remains unclear. Here we sought to identify the precise stage of erythropoiesis that depends on BCL-XL. We also tested whether deficiency of BIM or PUMA, the two main pro-apoptotic antagonists of BCL-XL, could prevent reticulocyte death and anaemia caused by BCL-XL loss. Using an in vivo mouse model of tamoxifen-inducible Bclx gene deletion and in vitro assays with a BCL-XL-selective inhibitor, we interrogated each stage of erythrocyte differentiation for BCL-XL dependency. This revealed that reticulocytes, but not orthochromatic erythroblasts, require BCL-XL for their survival. Surprisingly, concurrent loss of BIM or PUMA had no significant impact on the development of anemia following acute BCL-XL deletion in vivo. However, analysis of mixed bone marrow chimaeric mice revealed that loss of PUMA, but not loss of BIM, partially alleviated impaired erythropoiesis caused by BCL-XL deficiency. Insight into how the network of pro-survival and pro-apoptotic proteins works will assist the development of strategies to mitigate the effects of abnormal cell death during erythropoiesis and prevent anaemia in patients treated with BCL-XL-specific BH3-mimetic drugs.


Assuntos
Anemia/genética , Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Proteína 11 Semelhante a Bcl-2/genética , Regulação da Expressão Gênica , Proteínas Supressoras de Tumor/genética , Proteína bcl-X/genética , Anemia/metabolismo , Anemia/patologia , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteína 11 Semelhante a Bcl-2/deficiência , Benzotiazóis/farmacologia , Sobrevivência Celular , Modelos Animais de Doenças , Eritroblastos/metabolismo , Eritroblastos/patologia , Eritropoese/genética , Deleção de Genes , Humanos , Isoquinolinas/farmacologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Domínios Proteicos , Reticulócitos/metabolismo , Reticulócitos/patologia , Transdução de Sinais , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/deficiência , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/deficiência
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