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1.
Nat Commun ; 11(1): 4471, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901010

RESUMO

A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and division. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.


Assuntos
DNA Mitocondrial/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Transporte Biológico Ativo , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Cinesina/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteínas rho de Ligação ao GTP/metabolismo
2.
Nat Commun ; 11(1): 3388, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636396

RESUMO

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.


Assuntos
Corantes Fluorescentes/química , Hidrogéis/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Tampões (Química) , Células COS , Centríolos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlorocebus aethiops , Simulação por Computador , Eletrólitos , Epitopos , Imageamento Tridimensional , Microtúbulos/metabolismo , Distribuição Normal , Fotoquímica
3.
Nat Commun ; 11(1): 3298, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620747

RESUMO

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime, where the endolysosomal pathway plays a prominent role by regulating protein and lipid sorting and degradation. Here we report that TMEM16K, an endoplasmic reticulum lipid scramblase causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with the small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Anoctaminas/genética , Transporte Biológico , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Endossomos/ultraestrutura , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/ultraestrutura , Camundongos Knockout , Microscopia Eletrônica , Mutação , Transporte Proteico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
4.
Euro Surveill ; 25(28)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32700671

RESUMO

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Animais , Betacoronavirus/genética , Western Blotting , Células COS , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Genoma Viral , Camundongos , Pandemias , Peptidil Dipeptidase A/imunologia , Plasmídeos , Pneumonia Viral/genética , Proteínas Recombinantes/imunologia , Vírus da SARS/genética , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Transfecção , Células Vero , Integração Viral
5.
Ecotoxicol Environ Saf ; 201: 110835, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32563159

RESUMO

The activation of the aryl hydrocarbon receptor (AHR) occurs through the binding of dioxin-like compounds (DLCs) or natural ligands. In this pathway, the AHR-ARNT (AHR nuclear translocator) heterodimer serves to regulate critical physiological functions, such as immune responses and the metabolism of xenobiotics. Birds have three AHR isoforms (AHR1, AHR1ß, and AHR2) and two ARNT isoforms (ARNT1 and ARNT2). However, how AHR and ARNT dimerization pair in birds regulates the AHR signaling pathway in an isoform-specific manner remains unknown. In this study, we initially sought to clarify the major chicken AHR-ARNT (ckAHR-ckARNT) pairs by estimating the mRNA tissue distributions of various ckAHR and ckARNT isoforms. Our results indicated that the ckAHR1-ckARNT1 represented the major dimerization pair in most tissues except the brain. We then measured the transactivation potencies of various ckAHR-ckARNT pairs by natural ligands and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in in vitro reporter gene assays using COS-7 and LMH cell lines. Our results from the in vitro assays demonstrated that the ckAHR1-ckARNT1 pair was strongly activated by the five natural ligands, namely, 6-formylindolo [3,2-b]carbazole, L-kynurenin, kynurenic acid, indoxyl-3-sulfate, and 1,3,7-tribromodibenzo-p-dioxin, but not by TCDD. In in silico ligand docking simulations with ckAHR1 homology models, all the natural ligands showed a interaction pattern that was distinct from that observed with anthropogenic DLCs, including TCDD. In conclusion, our findings indicate that the ckAHR1-ckARNT1 may be the most important dimerization pair in most tissues for regulating the physiological functions driven by natural ligands, although it was less reactive to TCDD.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Galinhas/metabolismo , Dibenzodioxinas Policloradas/metabolismo , Multimerização Proteica , Receptores de Hidrocarboneto Arílico/metabolismo , Xenobióticos/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Simulação por Computador , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Isoformas de Proteínas , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Especificidade da Espécie , Transfecção
6.
Nat Commun ; 11(1): 2922, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523103

RESUMO

The conversion of white adipocytes to thermogenic beige adipocytes represents a potential mechanism to treat obesity and related metabolic disorders. However, the mechanisms involved in converting white to beige adipose tissue remain incompletely understood. Here we show profound beiging in a genetic mouse model lacking the transcriptional repressor Krüppel-like factor 3 (KLF3). Bone marrow transplants from these animals confer the beige phenotype on wild type recipients. Analysis of the cellular and molecular changes reveal an accumulation of eosinophils in adipose tissue. We examine the transcriptomic profile of adipose-resident eosinophils and posit that KLF3 regulates adipose tissue function via transcriptional control of secreted molecules linked to beiging. Furthermore, we provide evidence that eosinophils may directly act on adipocytes to drive beiging and highlight the critical role of these little-understood immune cells in thermogenesis.


Assuntos
Tecido Adiposo/metabolismo , Eosinófilos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Transdução de Sinais/fisiologia , Adiposidade/genética , Adiposidade/fisiologia , Animais , Células COS , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Citometria de Fluxo , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Obesidade/metabolismo , Transdução de Sinais/genética , Software
7.
Toxicol Appl Pharmacol ; 399: 115035, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32422327

RESUMO

BACKGROUND AND AIMS: Cholecystokinin (CCK) may potentially be used to treat obesity. However, it is well-known to induce acute pancreatitis and pancreas neoplasia in rodents, but not in primates. Here we report the nonclinical safety profile of a long-acting CCK-1 receptor (CCK-1R) agonist, NN9056, in rats and monkeys to support a First-in-Man clinical trial with NN9056. METHODS: Thirteen-week toxicological studies were conducted in rats and non-human primates followed by histopathological evaluation of affected tissues. NN9056 was characterised in vitro, and CCK-1R expression was assessed by in situ hybridization in cynomolgus monkey and human pancreas tissues. RESULTS: Affinity and potency of NN9056 was comparable to native sulphated CCK-8 (CCK-8) across species on the CCK-1R while it had no effect on the CCK-2 receptor (CCK-2R). In situ hybridization demonstrated abundant expression of CCK-1Rs in the exocrine pancreas of the rat. In contrast, it was only discreetly expressed on pancreatic acinar cells in the periphery of scattered lobules in monkeys. A similar expression pattern was observed in human pancreas. 13-weeks daily dosing with NN9056 produced the expected pancreatic pathological findings in rats. In monkeys, NN9056 increased pancreas weight and induced histopathological changes despite the low expression level of CCK-1Rs. CONCLUSION: Surprisingly, chronic CCK-1R activation constitutes a risk for pancreatitis and trophic actions on the exocrine pancreas in monkeys. Since similar CCK-1R expression patterns were found in pancreas of monkeys and humans this risk is likely translatable to humans and clinical development of NN9056 was therefore halted.


Assuntos
Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/patologia , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Receptores da Colecistocinina/agonistas , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Animais , Células COS , Chlorocebus aethiops , Colecistocinina/metabolismo , Humanos , Macaca fascicularis , Primatas , Ratos
8.
Mol Genet Genomics ; 295(5): 1103-1111, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32435981

RESUMO

The androgen insensitivity syndrome (AIS) is a congenital disease characterized by androgen resistance due to androgen receptor (AR) gene mutations, resulting in disorders of sex differentiation in 46,XY individuals. However, the underlying mechanisms in the majority of AR variants and the phenotype-genotype correlations are unclear. Here, we identified a p.Y764H variant of the AR gene that results in different phenotypes in a family. Structural analyses revealed that amino acid substitution affected protein properties and spatial conformation, and in vitro, functional studies showed impaired nuclear translocation ability of the mutated protein. Moreover, the extent to which this variant reduced nuclear translocation depends on the dihydrotestosterone (DHT) concentrations. Our results, for the first time, demonstrated a pathogenesis of the p.Y764H mutations in AR resulting in AIS phenotype, and indicated that AIS patients with p.Y764H mutation and preserved gonad might have residual AR activity at high androgen levels, putting patients at risk for pubertal virilization in the future. We provide an in-depth insight into the pathogenesis in AIS based on the amino acid substitution, which may help aid its precise diagnosis, personalized treatment, and organized follow-up to avoid gender dysphoria.


Assuntos
Substituição de Aminoácidos , Síndrome de Resistência a Andrógenos/genética , Núcleo Celular/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Sequenciamento Completo do Exoma/métodos , Síndrome de Resistência a Andrógenos/metabolismo , Animais , Células COS , China , Chlorocebus aethiops , Feminino , Hemizigoto , Humanos , Lactente , Masculino , Linhagem , Fenótipo , Transporte Proteico , Receptores Androgênicos/química , Irmãos
9.
Nat Commun ; 11(1): 2266, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385244

RESUMO

The evolutionarily conserved Par3/Par6/aPKC complex regulates the polarity establishment of diverse cell types and distinct polarity-driven functions. However, how the Par complex is concentrated beneath the membrane to initiate cell polarization remains unclear. Here we show that the Par complex exhibits cell cycle-dependent condensation in Drosophila neuroblasts, driven by liquid-liquid phase separation. The open conformation of Par3 undergoes autonomous phase separation likely due to its NTD-mediated oligomerization. Par6, via C-terminal tail binding to Par3 PDZ3, can be enriched to Par3 condensates and in return dramatically promote Par3 phase separation. aPKC can also be concentrated to the Par3N/Par6 condensates as a client. Interestingly, activated aPKC can disperse the Par3/Par6 condensates via phosphorylation of Par3. Perturbations of Par3/Par6 phase separation impair the establishment of apical-basal polarity during neuroblast asymmetric divisions and lead to defective lineage development. We propose that phase separation may be a common mechanism for localized cortical condensation of cell polarity complexes.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Células COS , Ciclo Celular , Diferenciação Celular , Sobrevivência Celular , Chlorocebus aethiops , Proteínas de Drosophila/química , Drosophila melanogaster/citologia , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Larva/citologia , Complexos Multiproteicos/química , Neurônios/citologia , Neurônios/metabolismo , Domínios Proteicos , Proteína Quinase C/metabolismo , Ratos
10.
Nucleic Acids Res ; 48(9): 4658-4671, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32313951

RESUMO

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.


Assuntos
Ferroquelatase/genética , Oligonucleotídeos/administração & dosagem , Protoporfiria Eritropoética/metabolismo , Processamento de RNA , Albuminas/metabolismo , Animais , Medula Óssea/metabolismo , Células COS , Chlorocebus aethiops , Modelos Animais de Doenças , Ferroquelatase/metabolismo , Humanos , Células K562 , Camundongos , Oligonucleotídeos/sangue , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Polimorfismo de Nucleotídeo Único , Protoporfiria Eritropoética/genética , Protoporfiria Eritropoética/terapia , Sítios de Splice de RNA , Distribuição Tecidual
11.
PLoS One ; 15(4): e0231750, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324760

RESUMO

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1+/+ but not on Trpm1-/- mouse retina as shown by co-immunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry). Therefore, we propose that a combination of different methods should be used to test for the presence of anti-TRPM1 autoantibodies in the sera of MAR patients.


Assuntos
Autoanticorpos/sangue , Melanoma/imunologia , Síndromes Paraneoplásicas Oculares/imunologia , Retina/imunologia , Doenças Retinianas/imunologia , Canais de Cátion TRPM/imunologia , Idoso , Animais , Células COS , Chlorocebus aethiops , Feminino , Humanos , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Retina/patologia
12.
Proc Natl Acad Sci U S A ; 117(19): 10378-10387, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332162

RESUMO

Barrier-to-autointegration factor (BAF) is a highly conserved protein in metazoans that has multiple functions during the cell cycle. We found that BAF is SUMOylated at K6, and that this modification is essential for its nuclear localization and function, including nuclear integrity maintenance and DNA replication. K6-linked SUMOylation of BAF promotes binding and interaction with lamin A/C to regulate nuclear integrity. K6-linked SUMOylation of BAF also supports BAF binding to DNA and proliferating cell nuclear antigen and regulates DNA replication. SENP1 and SENP2 catalyze the de-SUMOylation of BAF at K6. Disrupting the SUMOylation and de-SUMOylation cycle of BAF at K6 not only disturbs nuclear integrity, but also induces DNA replication failure. Taken together, our findings demonstrate that SUMOylation at K6 is an important regulatory mechanism that governs the nuclear functions of BAF in mammalian cells.


Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Lamina Tipo A/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Sinais de Localização Nuclear/genética , Proteínas Nucleares/metabolismo , Ligação Proteica/fisiologia , Sumoilação/fisiologia
13.
Nat Commun ; 11(1): 1953, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327662

RESUMO

Plexins are receptors for semaphorins that transduce signals for regulating neuronal development and other processes. Plexins are single-pass transmembrane proteins with multiple domains in both the extracellular and intracellular regions. Semaphorin activates plexin by binding to its extracellular N-terminal Sema domain, inducing the active dimer of the plexin intracellular region. The mechanism underlying this activation process of plexin is incompletely understood. We present cryo-electron microscopic structure of full-length human PlexinC1 in complex with the viral semaphorin mimic A39R. The structure shows that A39R induces a specific dimer of PlexinC1 where the membrane-proximal domains from the two PlexinC1 protomers are placed close to each other, poised to promote the active dimer of the intracellular region. This configuration is imposed by a distinct conformation of the PlexinC1 extracellular region, stabilized by inter-domain interactions among the Sema and membrane-proximal domains. Our mutational analyses support the critical role of this conformation in PlexinC1 activation.


Assuntos
Receptores Virais/química , Receptores Virais/metabolismo , Semaforinas/química , Semaforinas/metabolismo , Transdução de Sinais , Animais , Células COS , Chlorocebus aethiops , Microscopia Crioeletrônica , Humanos , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Virais/genética , Relação Estrutura-Atividade
14.
Nat Cell Biol ; 22(4): 498-511, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203420

RESUMO

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.


Assuntos
Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Integrinas/genética , Mecanotransdução Celular/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células COS , Adesão Celular , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Biologia Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Cães , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Pan troglodytes , Domínios Proteicos , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/classificação , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
15.
Nat Commun ; 11(1): 1127, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111841

RESUMO

Although viruses must navigate the complex host endomembrane system to infect cells, the strategies used to achieve this is unclear. During entry, polyomavirus SV40 is sorted from the late endosome (LE) to the endoplasmic reticulum (ER) to cause infection, yet how this is accomplished remains enigmatic. Here we find that EMC4 and EMC7, two ER membrane protein complex (EMC) subunits, support SV40 infection by promoting LE-to-ER targeting of the virus. They do this by engaging LE-associated Rab7, presumably to stabilize contact between the LE and ER. These EMC subunits also bind to the ER-resident fusion machinery component syntaxin18, which is required for SV40-arrival to the ER. Our data suggest that EMC4 and EMC7 act as molecular tethers, inter-connecting two intracellular compartments to enable efficient transport of a virus between these compartments. As LE-to-ER transport of cellular cargos is unclear, our results have broad implications for illuminating inter-organelle cargo transport.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Internalização do Vírus , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Membranas Intracelulares/virologia , Proteínas de Membrana/genética , Ligação Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Vírus 40 dos Símios/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
16.
Nat Chem Biol ; 16(4): 400-407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32198492

RESUMO

The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we present a 1.8 Å-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target.


Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Fosfolipase D/ultraestrutura , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células COS , Catálise , Domínio Catalítico , Chlorocebus aethiops , Humanos , Lipídeos de Membrana , Fosfatidilcolinas , Ligação Proteica , Proteína Quinase C/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais/efeitos dos fármacos
17.
Science ; 367(6484): 1366-1371, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193326

RESUMO

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIß [PI(4)KIIIß], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission.


Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Fosfatos de Fosfatidilinositol/metabolismo , Rede trans-Golgi/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Dinaminas/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Microdomínios da Membrana , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Interferência de RNA
18.
Nat Commun ; 11(1): 1489, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198364

RESUMO

Axon pathfinding is critical for nervous system development, and it is orchestrated by molecular cues that activate receptors on the axonal growth cone. Robo family receptors bind Slit guidance cues to mediate axon repulsion. In mammals, the divergent family member Robo3 does not bind Slits, but instead signals axon repulsion from its own ligand, NELL2. Conversely, canonical Robos do not mediate NELL2 signaling. Here, we present the structures of NELL-Robo3 complexes, identifying a mode of ligand engagement for Robos that is orthogonal to Slit binding. We elucidate the structural basis for differential binding between NELL and Robo family members and show that NELL2 repulsive activity is a function of its Robo3 affinity and is enhanced by ligand trimerization. Our results reveal a mechanism of oligomerization-induced Robo activation for axon guidance and shed light on Robo family member ligand binding specificity, conformational variability, divergent modes of signaling, and evolution.


Assuntos
Orientação de Axônios/fisiologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Animais , Axônios/metabolismo , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Drosophila , Proteínas de Drosophila/metabolismo , Mamíferos , Camundongos , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Espalhamento de Radiação , Transdução de Sinais
19.
Proc Natl Acad Sci U S A ; 117(13): 7225-7235, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32179693

RESUMO

The endoplasmic reticulum (ER) is the site of synthesis of secretory and membrane proteins and contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner. How the ER spatially segregates its numerous and diverse functions, including positioning nanoscopic contact sites with other organelles, is unclear. We demonstrate that hypotonic swelling of cells converts the ER and other membrane-bound organelles into micrometer-scale large intracellular vesicles (LICVs) that retain luminal protein content and maintain contact sites with each other through localized organelle tethers. Upon cooling, ER-derived LICVs phase-partition into microscopic domains having different lipid-ordering characteristics, which is reversible upon warming. Ordered ER lipid domains mark contact sites with ER and mitochondria, lipid droplets, endosomes, or plasma membrane, whereas disordered ER lipid domains mark contact sites with lysosomes or peroxisomes. Tethering proteins concentrate at ER-organelle contact sites, allowing time-dependent behavior of lipids and proteins to be studied at these sites. These findings demonstrate that LICVs provide a useful model system for studying the phase behavior and interactive properties of organelles in intact cells.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/fisiologia , Membranas Mitocondriais/metabolismo , Animais , Transporte Biológico , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células HEK293 , Humanos , Lipídeos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Transporte Proteico
20.
Nucleic Acids Res ; 48(8): 4028-4040, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32170309

RESUMO

In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification.


Assuntos
Acetilgalactosamina/química , Carboidratos/química , Interferência de RNA , RNA Interferente Pequeno/química , Álcoois Açúcares/química , Animais , Células COS , Chlorocebus aethiops , Exorribonucleases , Hepatócitos/metabolismo , Camundongos , Conformação de Ácido Nucleico , Pré-Albumina/genética , Ribonucleotídeos/química
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