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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): 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
3.
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
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.
Nat Methods ; 17(7): 726-733, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572233

RESUMO

Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm2 while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.


Assuntos
Microscopia de Fluorescência/métodos , Animais , Células COS , Centríolos/ultraestrutura , Chlorocebus aethiops , Humanos , Imageamento Tridimensional , Iluminação , Microtúbulos/ultraestrutura
8.
Proc Natl Acad Sci U S A ; 117(26): 14978-14986, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554490

RESUMO

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.


Assuntos
Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , Receptores de Superfície Celular/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Receptores de Superfície Celular/genética
9.
Invest Ophthalmol Vis Sci ; 61(5): 41, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32446246

RESUMO

Purpose: To identify the pathogenic gene of infantile nystagmus syndrome (INS) in three Chinese families and explore the potential pathogenic mechanism of FERM domain-containing 7 (FRMD7) mutations. Methods: Genetic testing was performed via Sanger sequencing. Western blotting was used to analyze protein expression of FRMD7. Glutathione S-transferase pull-down and immunoprecipitation were conducted to investigate the proteins interacting with FRMD7. Rescue assays were performed in Caenorhabditis elegans to explore the potential role of FRMD7 in vivo. Results: We recruited three Chinese families with X-linked INS and identified a duplication and two missense mutations in FRMD7: c.998dupA/p.His333Glnfs*2, c.580G>A/p.Ala194Thr, and c.973A>G/p.Arg325Gly (one in each family). Expression levels of three mutants were similar to that of wild-type FRMD7 in vitro. Interestingly, the mutant p.His333Glnfs*2 exhibited a predominantly nuclear location, whereas wild-type FRMD7 localized to the cytoplasm. In addition, we found FRMD7 to directly interact with the loop between transmembrane domains 3 and 4 of GABRA2, a type A gamma-aminobutyric acid (GABA) receptor (GABAARs) subunit critical for receptor transport and localization, whereas the mutants p.Ala194Thr and p.Arg325Gly exhibited decreased binding to GABRA2. In frm-3 (a nematode homologue of FRMD7) null C. elegans, we found that FRMD7 mutants exhibited a poor rescue effect on the defects of locomotion and fluorescence recovery after photobleaching of GABAARs. Conclusions: Our findings identified three FRMD7 mutants in three Chinese families with X-linked INS and confirmed GABRA2 as a novel binding partner of FRMD7. These findings suggest that FRMD7 plays an important role by targeting GABAARs.


Assuntos
Proteínas do Citoesqueleto/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteínas de Membrana/genética , Mutação , Nistagmo Congênito/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de GABA-A/metabolismo , Animais , Grupo com Ancestrais do Continente Asiático/genética , Western Blotting , Células COS , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , China/epidemiologia , Chlorocebus aethiops , Proteínas do Citoesqueleto/metabolismo , Análise Mutacional de DNA , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Testes Genéticos , Humanos , Imunoprecipitação , Masculino , Proteínas de Membrana/metabolismo , Nistagmo Congênito/metabolismo , Linhagem , Plasmídeos/genética
10.
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
11.
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
12.
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
13.
Am J Physiol Renal Physiol ; 319(1): F52-F62, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32463725

RESUMO

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.


Assuntos
Proteínas 14-3-3/metabolismo , Rim/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Animais , Butadienos/farmacologia , Células COS , Chlorocebus aethiops , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Nitrilos/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
J Neurosci ; 40(23): 4609-4619, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32350039

RESUMO

Idebenone is a synthetic quinone that on reduction in cells can bypass mitochondrial Complex I defects by donating electrons to Complex III. The drug is used clinically to treat the Complex I disease Leber's hereditary optic neuropathy (LHON), but has been less successful in clinical trials for other neurodegenerative diseases. NAD(P)H:quinone oxidoreductase 1 (NQO1) appears to be the main intracellular enzyme catalyzing idebenone reduction. However, NQO1 is not universally expressed by cells of the brain. Using primary rat cortical cells pooled from both sexes, we tested the hypotheses that the level of endogenous NQO1 activity limits the ability of neurons, but not astrocytes, to use idebenone as an electron donor to support mitochondrial respiration. We then tested the prediction that NQO1 induction by pharmacological activation of the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) enables idebenone to bypass Complex I in cells with poor NQO1 expression. We found that idebenone stimulated respiration by astrocytes but reduced the respiratory capacity of neurons. Importantly, idebenone supported mitochondrial oxygen consumption in the presence of a Complex I inhibitor in astrocytes but not neurons, and this ability was reversed by inhibiting NQO1. Conversely, recombinant NQO1 delivery to neurons prevented respiratory impairment and conferred Complex I bypass activity. Nrf2 activators failed to increase NQO1 in neurons, but carnosic acid induced NQO1 in COS-7 cells that expressed little endogenous enzyme. Carnosic acid-idebenone combination treatment promoted NQO1-dependent Complex I bypass activity in these cells. Thus, combination drug strategies targeting NQO1 may promote the repurposing of idebenone for additional disorders.SIGNIFICANCE STATEMENT Idebenone is used clinically to treat loss of visual acuity in Leber's hereditary optic neuropathy. Clinical trials for several additional diseases have failed. This study demonstrates a fundamental difference in the way idebenone affects mitochondrial respiration in cortical neurons compared with cortical astrocytes. Cortical neurons are unable to use idebenone as a direct mitochondrial electron donor due to NQO1 deficiency. Our results suggest that idebenone behaves as an NQO1-dependent prodrug, raising the possibility that lack of neuronal NQO1 activity has contributed to the limited efficacy of idebenone in neurodegenerative disease treatment. Combination therapy with drugs able to safely induce NQO1 in neurons, as well as other brain cell types, may be able to unlock the neuroprotective therapeutic potential of idebenone or related quinones.


Assuntos
Antioxidantes/farmacologia , Astrócitos/enzimologia , Respiração Celular/fisiologia , Mitocôndrias/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ubiquinona/análogos & derivados , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células COS , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Masculino , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ubiquinona/farmacologia
15.
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
16.
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
17.
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
18.
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
19.
J Neurosci ; 40(19): 3720-3740, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32273484

RESUMO

Nestin, an intermediate filament protein widely used as a marker of neural progenitors, was recently found to be expressed transiently in developing cortical neurons in culture and in developing mouse cortex. In young cortical cultures, nestin regulates axonal growth cone morphology. In addition, nestin, which is known to bind the neuronal cdk5/p35 kinase, affects responses to axon guidance cues upstream of cdk5, specifically, to Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, and changes in microtubules and actin filaments are well studied. In contrast, the roles of intermediate filament proteins in this process are poorly understood, even in cultured neurons. Here, we investigate the molecular mechanism by which nestin affects growth cone morphology and Sema3a sensitivity. We find that nestin selectively facilitates the phosphorylation of the lissencephaly-linked protein doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected by nestin. We uncover that this substrate selectivity is based on the ability of nestin to interact with DCX, but not with other cdk5 substrates. Nestin thus creates a selective scaffold for DCX with activated cdk5/p35. Last, we use cortical cultures derived from Dcx KO mice to show that the effects of nestin on growth cone morphology and on Sema3a sensitivity are DCX-dependent, thus suggesting a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating the intracellular kinase signaling environment in developing neurons. The sex of animal subjects is unknown.SIGNIFICANCE STATEMENT Nestin, an intermediate filament protein highly expressed in neural progenitors, was recently identified in developing neurons where it regulates growth cone morphology and responsiveness to the guidance cue Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, but the roles of intermediate filaments in this process are poorly understood. We now report that nestin selectively facilitates phosphorylation of the lissencephaly-linked doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected. This substrate selectivity is based on preferential scaffolding of DCX, cdk5, and p35 by nestin. Additionally, we demonstrate a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating intracellular kinase signaling in developing neurons.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Nestina/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Feminino , Cones de Crescimento/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Semaforina-3A/metabolismo
20.
Genes Cells ; 25(6): 375-390, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32181976

RESUMO

PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5' Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-ß stimulation in breast cancer cells. We then generated CRISPR/Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Proteínas Smad/metabolismo , Motivos de Aminoácidos , Animais , Neoplasias da Mama/genética , Células COS , Carcinogênese/genética , Proliferação de Células/genética , Chlorocebus aethiops , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Organoides/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Smad/genética , Fator de Crescimento Transformador beta/farmacologia
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