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3.
Nat Med ; 25(12): 1839-1842, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31768065

RESUMO

Histiocytoses are clonal hematopoietic disorders frequently driven by mutations mapping to the BRAF and MEK1 and MEK2 kinases. Currently, however, the developmental origins of histiocytoses in patients are not well understood, and clinically meaningful therapeutic targets outside of BRAF and MEK are undefined. In this study, we uncovered activating mutations in CSF1R and rearrangements in RET and ALK that conferred dramatic responses to selective inhibition of RET (selpercatinib) and crizotinib, respectively, in patients with histiocytosis.


Assuntos
Quinase do Linfoma Anaplásico/genética , Histiocitose/genética , Proteínas Proto-Oncogênicas c-ret/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Adolescente , Adulto , Aminopiridinas/farmacologia , Benzotiazóis/farmacologia , Criança , Pré-Escolar , Feminino , Genoma Humano , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Histiocitose/tratamento farmacológico , Histiocitose/patologia , Humanos , Lactente , Masculino , Mutação , Ácidos Picolínicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Receptores Proteína Tirosina Quinases/genética , Gêmeos Monozigóticos , Sequenciamento Completo do Exoma , Adulto Jovem
4.
Science ; 364(6442)2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31123109

RESUMO

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Asma/terapia , Glicoproteínas/química , Glicoproteínas/farmacologia , Imunidade Inata/efeitos dos fármacos , Lisofosfolipase/química , Lisofosfolipase/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Asma/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/terapia , Cristalização , Modelos Animais de Doenças , Glicoproteínas/administração & dosagem , Glicoproteínas/imunologia , Células Caliciformes/imunologia , Células Caliciformes/patologia , Humanos , Epitopos Imunodominantes/imunologia , Imunoglobulina E/imunologia , Lisofosfolipase/administração & dosagem , Lisofosfolipase/imunologia , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Muco/imunologia
5.
Nat Chem Biol ; 15(6): 641-649, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31011214

RESUMO

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.


Assuntos
Derivados de Benzeno/farmacologia , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Arabidopsis , Derivados de Benzeno/química , Cadeias Pesadas de Clatrina/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Tiofenos/farmacologia
6.
Nat Commun ; 10(1): 1729, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988283

RESUMO

RIPK1 regulates cell death and inflammation through kinase-dependent and -independent mechanisms. As a scaffold, RIPK1 inhibits caspase-8-dependent apoptosis and RIPK3/MLKL-dependent necroptosis. As a kinase, RIPK1 paradoxically induces these cell death modalities. The molecular switch between RIPK1 pro-survival and pro-death functions remains poorly understood. We identify phosphorylation of RIPK1 on Ser25 by IKKs as a key mechanism directly inhibiting RIPK1 kinase activity and preventing TNF-mediated RIPK1-dependent cell death. Mimicking Ser25 phosphorylation (S > D mutation) protects cells and mice from the cytotoxic effect of TNF in conditions of IKK inhibition. In line with their roles in IKK activation, TNF-induced Ser25 phosphorylation of RIPK1 is defective in TAK1- or SHARPIN-deficient cells and restoring phosphorylation protects these cells from TNF-induced death. Importantly, mimicking Ser25 phosphorylation compromises the in vivo cell death-dependent immune control of Yersinia infection, a physiological model of TAK1/IKK inhibition, and rescues the cell death-induced multi-organ inflammatory phenotype of the SHARPIN-deficient mice.


Assuntos
Apoptose , Modelos Imunológicos , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Caspase 8/genética , Caspase 8/metabolismo , Caspase 8/fisiologia , Linhagem Celular , Quinase I-kappa B/metabolismo , Quinase I-kappa B/fisiologia , Imunidade/fisiologia , Camundongos , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Serina/química , Serina/metabolismo , Yersinia , Yersiniose/imunologia
7.
Nat Commun ; 10(1): 1834, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015422

RESUMO

Prevention of inflammatory bowel disease (IBD) relies on tight control of inflammatory, cell death and autophagic mechanisms, but how these pathways are integrated at the molecular level is still unclear. Here we show that the anti-inflammatory protein A20 and the critical autophagic mediator Atg16l1 physically interact and synergize to regulate the stability of the intestinal epithelial barrier. A proteomic screen using the WD40 domain of ATG16L1 (WDD) identified A20 as a WDD-interacting protein. Loss of A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology, as characterized by severe inflammation and increased intestinal epithelial cell death in both small and large intestine. Mechanistically, absence of A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or expression of WDD-deficient Atg16l1 stabilizes A20. Collectively our data show that A20 and Atg16l1 cooperatively control intestinal homeostasis by acting at the intersection of inflammatory, autophagy and cell death pathways.


Assuntos
Proteínas de Transporte/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Repetições WD40/genética , Animais , Autofagia/imunologia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Endoscopia , Feminino , Homeostase/imunologia , Humanos , Doenças Inflamatórias Intestinais/diagnóstico por imagem , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/imunologia , Proteômica , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Repetições WD40/imunologia
8.
Nature ; 568(7753): 571-575, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30944476

RESUMO

Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8-11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.


Assuntos
ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/metabolismo , Ciclo do Ácido Cítrico , Evolução Molecular , ATP Citrato (pro-S)-Liase/genética , Biocatálise , Chlorobium/enzimologia , Chlorobium/genética , Cristalografia por Raios X , Humanos , Methanosarcinales/enzimologia , Methanosarcinales/genética , Modelos Moleculares
9.
J Allergy Clin Immunol ; 144(1): 204-215, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30876911

RESUMO

BACKGROUND: The emergence of IL-33 as a key molecular player in the development and propagation of widespread inflammatory diseases, including asthma and atopic dermatitis, has established the need for effective IL-33-neutralizing biologics. OBJECTIVE: Here we describe the development and validation of a new antagonist of IL-33, termed IL-33trap, which combines the extracellular domains of the IL-33 receptor (ST2) and its coreceptor, IL-1 receptor accessory protein, into a single fusion protein. METHODS: We produced and purified recombinant IL-33trap from human cells and analyzed its IL-33-binding affinity and IL-33 antagonistic activity in cultured cells and mice. IL-33trap activity was also benchmarked with a recombinant soluble ST2 corresponding to the naturally occurring IL-33 decoy receptor. Finally, we studied the effect of IL-33trap in the Alternaria alternata mouse model of allergic airway inflammation. RESULTS: In vitro IL-33trap binds IL-33 and inhibits IL-33 activity to a much stronger degree than soluble ST2. Furthermore, IL-33trap inhibits eosinophil infiltration, splenomegaly, and production of signature cytokines in splenic lymphocytes and lung tissue on IL-33 injection. Finally, administration of IL-33trap at the time of allergen challenge inhibits inflammatory responses in a preclinical mouse model of acute allergic airway inflammation. CONCLUSIONS: IL-33trap is a novel IL-33 antagonist that outperforms the natural IL-33 decoy receptor and shows anti-inflammatory activities in a preclinical mouse model of acute allergic airway inflammation when administered at the time of allergen challenge.

10.
Biochem Pharmacol ; 165: 240-248, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30885765

RESUMO

The four core members of the Interleukin-12 (IL-12) family of cytokines, IL-12, IL-23, IL-27 and IL-35 are heterodimers which share α- and ß-cytokine subunits. All four cytokines are immune modulators and have been proposed to play divergent roles in inflammatory arthritis. In recent years additional combinations of α- and ß-cytokine subunits belonging to the IL-12 family have been proposed to form novel cytokines such as IL-39. However, the actual extent of the combinatorial potential of the cytokine subunits in the human IL-12 family is not known. Here, we identify several combinations of subunits that form secreted heterodimeric assemblies based on a systematic orthogonal approach. The heterodimers are detected in the conditioned media harvested from mammalian cell cultures transfected with unfused pairs of cytokine subunits. While certain previously reported subunit combinations could not be recapitulated, our approach showed robustly that all four of the canonical members could be secreted. Furthermore, we provide evidence for the interaction between Cytokine Receptor Like Factor 1 (CRLF1) and Interleukin-12 subunit alpha (p35). Similar to IL-27 and IL-35 this novel heterodimer is not abundantly secreted rendering isolation from the conditioned medium very challenging, unlike IL-12 and IL-23. Our findings set the stage for fine-tuning approaches towards the biochemical reconstitution of IL-12 family cytokines for biochemical, cellular, and structural studies.

11.
Clin Immunol ; 206: 15-22, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30196070

RESUMO

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis of unknown etiology, and currently the cellular and molecular interactions that dictate its pathogenesis remain elusive. A role of the interleukin-23 (IL-23)/IL-23R (IL-23 receptor) interaction in the development of psoriasis and PsA is well established. As IL-23 regulates the differentiation and activation of innate and adaptive immunity, it pertains to a very complex pathophysiology involving a plethora of effectors and transducers. In this review, we will discuss recent advances on the cellular and molecular pathophysiological mechanisms that regulate the initiation and progression of PsA as well as new therapeutic approaches for IL-23/IL-23R targeted therapeutics.

12.
Nat Commun ; 9(1): 5340, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30559399

RESUMO

Activated invariant natural killer T (iNKT) cells rapidly produce large amounts of cytokines, but how cytokine mRNAs are induced, stabilized and mobilized following iNKT activation is still unclear. Here we show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), links key cellular processes required for iNKT cell effector functions in specific iNKT subsets, in which TCR-dependent activation of IRE1α is associated with downstream activation of p38 MAPK and the stabilization of preformed cytokine mRNAs. Importantly, genetic deletion of IRE1α in iNKT cells reduces cytokine production and protects mice from oxazolone colitis. We therefore propose that an IRE1α-dependent signaling cascade couples constitutive cytokine mRNA expression to the rapid induction of cytokine secretion and effector functions in activated iNKT cells.


Assuntos
Citocinas/genética , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/genética , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Proteínas Serina-Treonina Quinases/genética , Animais , Células Cultivadas , Colite/genética , Deleção de Genes , Camundongos , Camundongos Knockout , Oxazolona/toxicidade , RNA Mensageiro/genética , Transdução de Sinais , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Sci Rep ; 8(1): 16760, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425318

RESUMO

The ability of bacteria to infect a host relies in part on the secretion of molecular virulence factors across the cell envelope. Pseudomonas aeruginosa, a ubiquitous environmental bacterium causing opportunistic infections in humans, employs the type II secretion system (T2SS) to transport effector proteins across its cellular envelope as part of a diverse array of virulence strategies. General secretory pathway protein L (GspL) is an essential inner-membrane component of the T2SS apparatus, and is thought to facilitate transduction of the energy from ATP hydrolysis in the cytoplasm to the periplasmic components of the system. However, our incomplete understanding of the assembly principles of the T2SS machinery prevents the mechanistic deconvolution of T2SS-mediated protein secretion. Here we show via two crystal structures that the periplasmic ferredoxin-like domain of GspL (GspLfld) is a dimer stabilized by hydrophobic interactions, and that this interface may allow significant interdomain plasticity. The general dimerization mode of GspLfld is shared with GspL from Vibrio parahaemolyticus suggesting a conserved oligomerization mode across the GspL family. Furthermore, we identified a tetrameric form of the complete periplasmic segment of GspL (GspLperi) which indicates that GspL may be able to adopt multiple oligomeric states as part of its dynamic role in the T2SS apparatus.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Multimerização Proteica , Pseudomonas aeruginosa/metabolismo , Sistemas de Secreção Tipo II/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Domínios Proteicos , Estrutura Quaternária de Proteína
14.
Front Immunol ; 9: 2366, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30429846

RESUMO

Background: Inherited CARD9 deficiency constitutes a primary immunodeficiency predisposing uniquely to chronic and invasive fungal infections. Certain mutations are shown to negatively impact CARD9 protein expression and/or NF-κB activation, but the underlying biochemical mechanism remains to be fully understood. Objectives: To investigate a possible founder origin of a known CARD9 R70W mutation in five families of Turkish origin. To explore the biochemical mechanism of immunodeficiency by R70W CARD9. Methods: We performed haplotype analysis using microsatellite markers and SNPs. We designed a model system exploiting a gain-of-function (GOF) CARD9 L213LI mutant that triggers constitutive NF-κB activation, analogous to an oncogenic CARD11 mutant, to study NF-κB signaling and signalosome formation. We performed reporter assays, immunoprecipitation and confocal imaging on HEK cells overexpressing different CARD9 variants. Results: We identified a common haplotype, thus providing evidence for a common Turkish founder. CARD9 R70W failed to activate NF-κB and abrogated NF-κB activation by WT CARD9 and by GOF CARD9. Notably, R70W CARD9 also exerted negative effects on NF-κB activation by CARD10, CARD11, and CARD14. Consistent with the NF-κB results, the R70W mutation prevented GOF CARD9 to pull down the signalosome partner proteins BCL10 and MALT1. This reflected into drastic reduction of BCL10 filamentous assemblies in a cellular context. Indeed, structural analysis revealed that position R70 in CARD9 maps at the putative interface between successive CARD domains in CARD9 filaments. Conclusions: The R70W mutation in CARD9 prevents NF-κB activation by inhibiting productive interactions with downstream BCL10 and MALT1, necessary for assembly of the filamentous CARD9-BCL10-MALT1 signalosome.


Assuntos
Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Efeito Fundador , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Mutação , NF-kappa B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Sinalização CARD/química , Linhagem Celular , Suscetibilidade a Doenças , Feminino , Mutação com Ganho de Função , Humanos , Masculino , Modelos Moleculares , Linhagem , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
15.
Science ; 362(6417): 952-956, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30361387

RESUMO

Transforming growth factor-ß1 (TGF-ß1) is one of very few cytokines produced in a latent form, requiring activation to exert any of its vastly diverse effects on development, immunity, and cancer. Regulatory T cells (Tregs) suppress immune cells within close proximity by activating latent TGF-ß1 presented by GARP (glycoprotein A repetitions predominant) to integrin αVß8 on their surface. We solved the crystal structure of GARP:latent TGF-ß1 bound to an antibody that stabilizes the complex and blocks release of active TGF-ß1. This finding reveals how GARP exploits an unusual medley of interactions, including fold complementation by the amino terminus of TGF-ß1, to chaperone and orient the cytokine for binding and activation by αVß8. Thus, this work further elucidates the mechanism of antibody-mediated blockade of TGF-ß1 activation and immunosuppression by Tregs.


Assuntos
Tolerância Imunológica , Proteínas de Membrana/química , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/química , Humanos , Ativação Linfocitária , Proteínas de Membrana/imunologia , Conformação Proteica em Folha beta , Dobramento de Proteína , Fator de Crescimento Transformador beta1/imunologia
16.
Plant Cell ; 30(10): 2573-2593, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30018157

RESUMO

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.


Assuntos
Arabidopsis/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brefeldina A/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transporte Proteico , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
17.
Cell Rep ; 23(7): 2026-2038, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29768202

RESUMO

The endoplasmic reticulum (ER) is a complex network of sheets and tubules that is continuously remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in atlastins (ATLs), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is unclear. Using electron microscopy (EM) volume reconstruction of transfected cells, neurons, and patient fibroblasts, we show that hereditary sensory and autonomic neuropathy (HSAN)-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity but are unable to promote membrane fusion, suggesting a defect in an intermediate step of the ATL3 functional cycle. Our data show that the effects of ATL3 mutations on ER network organization go beyond a loss of fusion and shed light on neuropathies caused by atlastin defects.


Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/genética , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação/genética , Animais , Células COS , Retículo Endoplasmático/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Fusão de Membrana , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Multimerização Proteica
18.
Sci Rep ; 8(1): 6673, 2018 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-29691449

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

19.
J Med Chem ; 61(7): 2753-2775, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29510037

RESUMO

In recent years, thymidylate kinase (TMPK), an enzyme indispensable for bacterial DNA biosynthesis, has been pursued for the development of new antibacterial agents including against Mycobacterium tuberculosis, the causative agent for the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous efforts toward the exploration of novel and potent Mycobacterium tuberculosis TMPK ( MtTMPK) inhibitors, and reported here the design of a novel series of non-nucleoside inhibitors of MtTMPK. The inhibitors display hitherto unexplored interactions in the active site of MtTMPK, offering new insights into structure-activity relationships. To investigate the discrepancy between enzyme inhibitory activity and the whole-cell activity, experiments with efflux pump inhibitors and efflux pump knockout mutants were performed. The minimum inhibitory concentrations of particular inhibitors increased significantly when determined for the efflux pump mmr knockout mutant, which partly explains the observed dissonance.


Assuntos
Antituberculosos/síntese química , Antituberculosos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Núcleosídeo-Fosfato Quinase/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Desenho de Drogas , Técnicas de Inativação de Genes , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Núcleosídeo-Fosfato Quinase/genética , Relação Estrutura-Atividade
20.
Immunity ; 48(1): 45-58.e6, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29287995

RESUMO

Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rß1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.


Assuntos
Subunidade beta 1 de Receptor de Interleucina-12/metabolismo , Interleucina-23/metabolismo , Receptores de Interleucina/metabolismo , Animais , Calorimetria/métodos , Linhagem Celular , Humanos , Interferometria/métodos , Subunidade p40 da Interleucina-12/metabolismo , Masculino , Camundongos , Ligação Proteica/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
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