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1.
Nature ; 591(7849): 275-280, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33442058

RESUMO

The innate immune regulator STING is a critical sensor of self- and pathogen-derived DNA. DNA sensing by STING leads to the induction of type-I interferons (IFN-I) and other cytokines, which promote immune-cell-mediated eradication of pathogens and neoplastic cells1,2. STING is also a robust driver of antitumour immunity, which has led to the development of STING activators and small-molecule agonists as adjuvants for cancer immunotherapy3. Pain, transmitted by peripheral nociceptive sensory neurons (nociceptors), also aids in host defence by alerting organisms to the presence of potentially damaging stimuli, including pathogens and cancer cells4,5. Here we demonstrate that STING is a critical regulator of nociception through IFN-I signalling in peripheral nociceptors. We show that mice lacking STING or IFN-I signalling exhibit hypersensitivity to nociceptive stimuli and heightened nociceptor excitability. Conversely, intrathecal activation of STING produces robust antinociception in mice and non-human primates. STING-mediated antinociception is governed by IFN-Is, which rapidly suppress excitability of mouse, monkey and human nociceptors. Our findings establish the STING-IFN-I signalling axis as a critical regulator of physiological nociception and a promising new target for treating chronic pain.


Assuntos
Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Nociceptividade/fisiologia , Dor/metabolismo , Células Receptoras Sensoriais/metabolismo , Analgesia , Animais , Feminino , Humanos , Interferon Tipo I/deficiência , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Macaca mulatta , Masculino , Proteínas de Membrana/agonistas , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Nociceptividade/efeitos dos fármacos , Transdução de Sinais
2.
Nat Commun ; 12(1): 690, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514739

RESUMO

Lamins and transmembrane proteins within the nuclear envelope regulate nuclear structure and chromatin organization. Nuclear envelope transmembrane protein 39 (Net39) is a muscle nuclear envelope protein whose functions in vivo have not been explored. We show that mice lacking Net39 succumb to severe myopathy and juvenile lethality, with concomitant disruption in nuclear integrity, chromatin accessibility, gene expression, and metabolism. These abnormalities resemble those of Emery-Dreifuss muscular dystrophy (EDMD), caused by mutations in A-type lamins (LMNA) and other genes, like Emerin (EMD). We observe that Net39 is downregulated in EDMD patients, implicating Net39 in the pathogenesis of this disorder. Our findings highlight the role of Net39 at the nuclear envelope in maintaining muscle chromatin organization, gene expression and function, and its potential contribution to the molecular etiology of EDMD.


Assuntos
Proteínas de Membrana/deficiência , Músculo Esquelético/patologia , Distrofia Muscular de Emery-Dreifuss/genética , Membrana Nuclear/patologia , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Lamina Tipo A/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/genética , Fosfatidato Fosfatase/genética , RNA-Seq , Estudos Retrospectivos
3.
Nature ; 585(7825): 420-425, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879486

RESUMO

The opsin family of G-protein-coupled receptors are used as light detectors in animals. Opsin 5 (also known as neuropsin or OPN5) is a highly conserved opsin that is sensitive to visible violet light1,2. In mice, OPN5 is a known photoreceptor in the retina3 and skin4 but is also expressed in the hypothalamic preoptic area (POA)5. Here we describe a light-sensing pathway in which POA neurons that express Opn5 regulate thermogenesis in brown adipose tissue (BAT). We show that Opn5 is expressed in glutamatergic warm-sensing POA neurons that receive synaptic input from several thermoregulatory nuclei. We further show that Opn5 POA neurons project to BAT and decrease its activity under chemogenetic stimulation. Opn5-null mice show overactive BAT, increased body temperature, and exaggerated thermogenesis when cold-challenged. Moreover, violet photostimulation during cold exposure acutely suppresses BAT temperature in wild-type mice but not in Opn5-null mice. Direct measurements of intracellular cAMP ex vivo show that Opn5 POA neurons increase cAMP when stimulated with violet light. This analysis thus identifies a violet light-sensitive deep brain photoreceptor that normally suppresses BAT thermogenesis.


Assuntos
Cor , Luz , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Neurônios/efeitos da radiação , Opsinas/metabolismo , Área Pré-Óptica/citologia , Termogênese/efeitos da radiação , Tecido Adiposo Marrom/inervação , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/efeitos da radiação , Animais , Temperatura Corporal , Temperatura Baixa , AMP Cíclico/metabolismo , Feminino , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Opsinas/deficiência , Opsinas/genética , Termogênese/genética
4.
Chem Biol Interact ; 330: 109251, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32888910

RESUMO

Cisplatin induces acute renal failure in humans and mice.Tubular apoptosis, necrosis and inflammation are the primary pathogenesis of cisplatin-induced acute kidney injury(AKI). We previously reported that the depletion of Numb from proximal tubules exacerbates tubular cells apoptosis in cisplatin-induced AKI, however, the role of Numb in tubular necrosis and renal inflammation in cisplatin-induced AKI remains unclear. A mouse model of AKI was produced by cisplatin intraperitoneally injection in mice from proximal tubule-specific depletion of Numb (PT-Nb-KO) and their wild-type littermates (PT-Nb-WT) respectively. Renal Numb expression was determined by Western blotting. Renal morphological damage was examined by hematoxylin and eosin staining (H&E staining). Tubular necrosis was evaluated by histological study and the protein level of renal Mixed lineage kinase domain-like protein (MLKL) which is a molecular marker of necrosis. Leukocyte infiltration and pro-inflammatory cytokines was determined by immunostaining and quantitative real-time PCR (qRT-PCR) respectively.The protein level of Numb was dramatically decreased in kidneys of PT-Nb-KO mice compared with PT-Nb-WT mice. After cisplatin injection, a significant increase of tubular injury score and the protein level of renal MLKL were detected in PT-Nb-KO mice compared with those in PT-Nb-WT. In addition, the number of F4/80-positve and CD3-positive cells, markers for macrophages and neutraphils respectively, showed significantly increased in kidneys from PT-Nb-KO mice compared with those in PT-Nb-WT mice. Consistently, the gene expression of pro-inflammatory cytokines including TNF-α and MCP-1 in the kidneys was higher in PT-Nb-KO mice than those in PT-Nb-WT mice. Numb play additional protective role in cisplatin-induced AKI through ameliorating tubular necrosis and renal inflammation besides attenuating cisplatin-induced tubular apoptosis.


Assuntos
Lesão Renal Aguda/patologia , Cisplatino/efeitos adversos , Inflamação/prevenção & controle , Proteínas de Membrana/fisiologia , Necrose/prevenção & controle , Proteínas do Tecido Nervoso/fisiologia , Animais , Contagem de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/etiologia , Túbulos Renais Proximais/patologia , Mastócitos , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Necrose/etiologia , Proteínas do Tecido Nervoso/deficiência , Neutrófilos , Proteínas Quinases/metabolismo
5.
Nature ; 584(7820): 252-256, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760004

RESUMO

A fundamental challenge in developing treatments for autism spectrum disorders is the heterogeneity of the condition. More than one hundred genetic mutations confer high risk for autism, with each individual mutation accounting for only a small fraction of cases1-3. Subsets of risk genes can be grouped into functionally related pathways, most prominently those involving synaptic proteins, translational regulation, and chromatin modifications. To attempt to minimize this genetic complexity, recent therapeutic strategies have focused on the neuropeptides oxytocin and vasopressin4-6, which regulate aspects of social behaviour in mammals7. However, it is unclear whether genetic risk factors predispose individuals to autism as a result of modifications to oxytocinergic signalling. Here we report that an autism-associated mutation in the synaptic adhesion molecule Nlgn3 results in impaired oxytocin signalling in dopaminergic neurons and in altered behavioural responses to social novelty tests in mice. Notably, loss of Nlgn3 is accompanied by a disruption of translation homeostasis in the ventral tegmental area. Treatment of Nlgn3-knockout mice with a new, highly specific, brain-penetrant inhibitor of MAP kinase-interacting kinases resets the translation of mRNA and restores oxytocin signalling and social novelty responses. Thus, this work identifies a convergence between the genetic autism risk factor Nlgn3, regulation of translation, and oxytocinergic signalling. Focusing on such common core plasticity elements might provide a pragmatic approach to overcoming the heterogeneity of autism. Ultimately, this would enable mechanism-based stratification of patient populations to increase the success of therapeutic interventions.


Assuntos
Transtorno Autístico/metabolismo , Transtorno Autístico/psicologia , Modelos Animais de Doenças , Ocitocina/metabolismo , Comportamento Social , Animais , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/efeitos dos fármacos
6.
Arterioscler Thromb Vasc Biol ; 40(8): 1918-1934, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32522006

RESUMO

OBJECTIVE: ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. CONCLUSIONS: This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.


Assuntos
Proteínas ADAM/fisiologia , Angiotensina II/farmacologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/etiologia , Proteínas de Membrana/fisiologia , Remodelação Vascular/efeitos dos fármacos , Proteínas ADAM/deficiência , Animais , Proliferação de Células , Células Cultivadas , Humanos , Inflamação/etiologia , Masculino , Proteínas de Membrana/deficiência , Camundongos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Trombospondina 1/análise , Vasoconstrição
7.
J Steroid Biochem Mol Biol ; 200: 105691, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32380235

RESUMO

Human steroid 5α-reductase 2 (SRD5A2) plays a determinative role in the masculinization of external genitalia. To date, approximately 114 different mutations of the SRD5A2 gene have been reported; however, little information is available about their impact on catalytic function or their three-dimensional (3D) structures. We determined the effect of point mutations on the testosterone-depend kinetic constants (Km,app and Vmax,app) and structural characteristics of SRD5A2 from Mexican patients with 46,XY-steroid 5α-reductase 2 deficiency. PCR-SSCP assays identified ten distinct gene variants and sequencing analysis identified missense mutations [p.V3I, p.S14R, p.A52T, p.F118L, p.R145W, p.R171S, p.L226P, p.F229S, p.S245Y, and p.A248V]. Mutations were re-created by site-directed mutagenesis and expressed in HEK293 cells. Functional studies demonstrated that 8 variants led to partial (Km,app = 0.16-2.6 µM; Vmax,app = 224-2640 pmol/mg P/min) or complete losses of activity compared to the wild-type enzyme (Km,app = 0.7 µM; Vmax,app = 4044 pmol/mg P/min). All the mutations were assessed using multiple software tools and the results predicted that all of the mutations were associated with disease or damage. Mapping mutations on the model of a 3D structure of SRD5A2 demonstrated alterations in contact sites with their proximal amino acids. Our data show that mutations affect the catalytic efficiency (Vmax/Km) or result in residual enzymatic activity, which could be due to erroneous interactions between amino acid residues, the substrate testosterone, or NADPH.


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Proteínas de Membrana/genética , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/química , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/deficiência , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Análise Mutacional de DNA , Células HEK293 , Humanos , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida
8.
Sci Rep ; 10(1): 8611, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451386

RESUMO

In the spinal cord, the axonal tracts with various caliber sizes are myelinated by oligodendrocytes and function as high-velocity ways for motor and sensory nerve signals. In some neurological disorders, such as multiple sclerosis, demyelination of small caliber axons is observed in the spinal cord. While type I/II oligodendrocytes among the four types are known to myelinate small diameter axons, their characteristics including identification of regulating molecules have not been understood yet. Here, we first found that in the wild-type mouse spinal cord, type I/II oligodendrocytes, positive for carbonic anhydrase II (CAII), were located in the corticospinal tract, fasciculus gracilis, and the inside part of ventral funiculus, in which small diameter axons existed. The type I/II oligodendrocytes started to appear between postnatal day (P) 7 and 11. We further analyzed the type I/II oligodendrocytes in the mutant mice, whose small diameter axons were hypomyelinated due to the deficiency of teneurin-4. In the teneurin-4 deficient mice, type I/II oligodendrocytes were significantly reduced, and the onset of the defect was at P11. Our results suggest that CAII-positive type I/II oligodendrocytes myelinate small caliber axons in the spinal cord and teneurin-4 is the responsible molecule for the generation of type I/II oligodendrocytes.


Assuntos
Proteínas de Membrana/metabolismo , Oligodendroglia/metabolismo , Medula Espinal/metabolismo , Animais , Axônios/metabolismo , Anidrase Carbônica II/metabolismo , Filamentos Intermediários/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Oligodendroglia/enzimologia , Tratos Piramidais/metabolismo , Medula Espinal/crescimento & desenvolvimento
9.
J Neurosci ; 40(23): 4457-4468, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32371604

RESUMO

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibular-induced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.SIGNIFICANCE STATEMENT We assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants revealed the presence of two distinct cell types, an upper layer of teardrop-shaped cells that rely on Tmc2a, and a lower layer of gourd-shaped cells that rely on Tmc1/2b.


Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Audição/fisiologia , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/genética , Proteínas de Peixe-Zebra/genética , Estimulação Acústica/métodos , Animais , Animais Geneticamente Modificados , Células Ciliadas Auditivas Internas/química , Proteínas de Membrana/análise , Proteínas de Membrana/deficiência , Peixe-Zebra , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/deficiência
10.
Nature ; 580(7801): 124-129, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238941

RESUMO

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.


Assuntos
Relógios Biológicos/fisiologia , Desenvolvimento Embrionário/fisiologia , Células-Tronco Pluripotentes/citologia , Somitos/citologia , Somitos/crescimento & desenvolvimento , Anormalidades Múltiplas/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Relógios Biológicos/genética , Desenvolvimento Embrionário/genética , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicosiltransferases/deficiência , Glicosiltransferases/genética , Hérnia Diafragmática/genética , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Fenótipo , Somitos/metabolismo , Fatores de Tempo
11.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32295905

RESUMO

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , RNA Mensageiro/imunologia , RNA Viral/imunologia , Receptores Tipo I de Interleucina-1/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Processamento Alternativo , Animais , Caspase 1/deficiência , Caspase 1/genética , Caspase 1/imunologia , Colo do Útero/imunologia , Colo do Útero/virologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Papillomaviridae/crescimento & desenvolvimento , Papillomaviridae/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , RNA Mensageiro/genética , RNA Viral/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/deficiência , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/imunologia , Receptores CCR6/deficiência , Receptores CCR6/genética , Receptores CCR6/imunologia , Receptores CXCR3/deficiência , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores Tipo I de Interleucina-1/deficiência , Receptores Tipo I de Interleucina-1/genética , Receptores de Interleucina-8B/deficiência , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/imunologia , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais , Vagina/imunologia , Vagina/virologia
12.
Biochim Biophys Acta Bioenerg ; 1861(8): 148202, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275929

RESUMO

Protein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexome profiling with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Biotinilação , Evolução Molecular , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , Ligação Proteica
13.
Am J Physiol Regul Integr Comp Physiol ; 318(5): R981-R996, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32186893

RESUMO

Selenoprotein S (Seps1) can be protective against oxidative, endoplasmic reticulum (ER), and inflammatory stress. Seps1 global knockout mice are less active, possess compromised fast muscle ex vivo strength, and, depending on context, heightened inflammation. Oxidative, ER, and inflammatory stress modulates contractile function; hence, our aim was to investigate the effects of Seps1 gene dose on exercise performance. Seps1-/- knockout, Seps1-/+ heterozygous, and wild-type mice were randomized to 3 days of incremental, high-intensity treadmill running or a sedentary control group. On day 4, the in situ contractile function of fast tibialis anterior (TA) muscles was determined. Seps1 reduction or deletion compromised exercise capacity, decreasing distance run. TA strength was also reduced. In sedentary Seps1-/- knockout mice, TA fatigability was greater than wild-type mice, and this was ameliorated with exercise. Whereas, in Seps1+/- heterozygous mice, exercise compromised TA endurance. These impairments in exercise capacity and TA contractile function were not associated with increased inflammation or a dysregulated redox state. Seps1 is highly expressed in muscle fibers and blood vessels. Interestingly, Nos1 and Vegfa mRNA transcripts were decreased in TA muscles from Seps1-/- knockout and Seps1-/+ heterozygous mice. Impaired exercise performance with Seps1 reduction or deletion cannot be attributed to heightened cellular stress, but it may potentially be mediated, in part, by the effects of Seps1 on the microvasculature.


Assuntos
Citocinas/sangue , Estresse do Retículo Endoplasmático , Tolerância ao Exercício , Mediadores da Inflamação/sangue , Contração Isométrica , Proteínas de Membrana/deficiência , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Estresse Oxidativo , Condicionamento Físico Animal , Selenoproteínas/deficiência , Animais , Citocinas/genética , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação , Fadiga Muscular , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/patologia , Força Muscular , Músculo Esquelético/patologia , Oxirredução , Estresse Oxidativo/genética , Corrida , Selenoproteínas/genética , Fatores de Tempo
14.
Sci Rep ; 10(1): 5115, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198470

RESUMO

Millions of individuals worldwide suffer from impaired vision, a condition with multiple origins that often impinge upon the light sensing cells of the retina, the photoreceptors, affecting their integrity. The molecular components contributing to this integrity are however not yet fully understood. Here we have asked whether Secreted Frizzled Related Protein 1 (SFRP1) may be one of such factors. SFRP1 has a context-dependent function as modulator of Wnt signalling or of the proteolytic activity of A Disintegrin And Metalloproteases (ADAM) 10, a main regulator of neural cell-cell communication. We report that in Sfrp1-/- mice, the outer limiting membrane (OLM) is discontinuous and the photoreceptors disorganized and more prone to light-induced damage. Sfrp1 loss significantly enhances the effect of the Rpe65Leu450Leu genetic variant -present in the mouse genetic background- which confers sensitivity to light-induced stress. These alterations worsen with age, affect visual function and are associated to an increased proteolysis of Protocadherin 21 (PCDH21), localized at the photoreceptor outer segment, and N-cadherin, an OLM component. We thus propose that SFRP1 contributes to photoreceptor fitness with a mechanism that involves the maintenance of OLM integrity. These conclusions are discussed in view of the broader implication of SFRP1 in neurodegeneration and aging.


Assuntos
Membrana Celular/patologia , Proteínas de Membrana/genética , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/patologia , Transtornos da Visão/patologia , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Caderinas/metabolismo , Comunicação Celular/genética , Luz/efeitos adversos , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos da Visão/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , cis-trans-Isomerases/genética
15.
Nat Commun ; 11(1): 1031, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098969

RESUMO

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans.


Assuntos
Endocitose/fisiologia , Mutação com Perda de Função , Linfopenia/genética , Proteínas de Membrana/deficiência , Linfócitos T/fisiologia , Animais , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular , Células Cultivadas , Feminino , Infecções por HIV/genética , HIV-1/patogenicidade , Humanos , Células Jurkat , Linfopenia/patologia , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Linhagem , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/virologia
16.
Nat Commun ; 11(1): 965, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075961

RESUMO

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.


Assuntos
Coração/fisiopatologia , Proteínas de Membrana/deficiência , Retículo Sarcoplasmático/patologia , Animais , Cálcio/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático , Técnicas de Inativação de Genes , Inativação Gênica , Coração/crescimento & desenvolvimento , Cardiopatias/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Peixe-Zebra
17.
Nat Commun ; 11(1): 970, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080200

RESUMO

Deregulation of mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is one of the most common inherited metabolic disorders, due to deficiency of the mitochondrial methylmalonyl-coenzyme A mutase (MMUT). How MMUT deficiency triggers cell damage remains unknown, preventing the development of disease-modifying therapies. Here we combine genetic and pharmacological approaches to demonstrate that MMUT deficiency induces metabolic and mitochondrial alterations that are exacerbated by anomalies in PINK1/Parkin-mediated mitophagy, causing the accumulation of dysfunctional mitochondria that trigger epithelial stress and ultimately cell damage. Using drug-disease network perturbation modelling, we predict targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient-derived cells and alleviate phenotype changes in mmut-deficient zebrafish. These results suggest a link between primary MMUT deficiency, diseased mitochondria, mitophagy dysfunction and epithelial stress, and provide potential therapeutic perspectives for MMA.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Metilmalonil-CoA Mutase/deficiência , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Mitofagia/fisiologia , Alquil e Aril Transferases/deficiência , Alquil e Aril Transferases/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Erros Inatos do Metabolismo/genética , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Camundongos Knockout , Doenças Mitocondriais/genética , Mitofagia/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estresse Fisiológico , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Peixe-Zebra
18.
Int J Mol Sci ; 21(3)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013259

RESUMO

The important physiologic role of peroxisomes is shown by the occurrence of peroxisomal biogenesis disorders (PBDs) in humans. This spectrum of autosomal recessive metabolic disorders is characterized by defective peroxisome assembly and impaired peroxisomal functions. PBDs are caused by mutations in the peroxisomal biogenesis factors, which are required for the correct compartmentalization of peroxisomal matrix enzymes. Recent work from patient cells that contain the Pex1(G843D) point mutant suggested that the inhibition of the lysosome, and therefore the block of pexophagy, was beneficial for peroxisomal function. The resulting working model proposed that Pex1 may not be essential for matrix protein import at all, but rather for the prevention of pexophagy. Thus, the observed matrix protein import defect would not be caused by a lack of Pex1 activity, but rather by enhanced removal of peroxisomal membranes via pexophagy. In the present study, we can show that the specific block of PEX1 deletion-induced pexophagy does not restore peroxisomal matrix protein import or the peroxisomal function in beta-oxidation in yeast. Therefore, we conclude that Pex1 is directly and essentially involved in peroxisomal matrix protein import, and that the PEX1 deletion-induced pexophagy is not responsible for the defect in peroxisomal function. In order to point out the conserved mechanism, we discuss our findings in the context of the working models of peroxisomal biogenesis and pexophagy in yeasts and mammals.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Membrana/genética , Sinais de Orientação para Peroxissomos/genética , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/deficiência , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Macroautofagia , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Peroxinas/genética , Peroxinas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Nature ; 579(7799): 443-447, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32103179

RESUMO

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Biocatálise , Domínio Catalítico , Sequência Conservada , Dolicol Monofosfato Manose/metabolismo , Fosfatos de Dolicol/metabolismo , Glucose/análogos & derivados , Glucose/metabolismo , Glicosiltransferases/deficiência , Técnicas In Vitro , Lipídeos , Proteínas de Membrana/deficiência , Modelos Moleculares , Mutação , Monossacarídeos de Poli-Isoprenil Fosfato/química , Monossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Especificidade por Substrato
20.
Am J Physiol Cell Physiol ; 318(3): C675-C694, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913700

RESUMO

Septate junctions (SJs) are occluding cell-cell junctions that have roles in paracellular permeability and barrier function in the epithelia of invertebrates. Arthropods have two types of SJs, pleated SJs and smooth SJs (sSJs). In Drosophila melanogaster, sSJs are found in the midgut and Malpighian tubules, but the functions of sSJs and their protein components in the tubule epithelium are unknown. Here we examined the role of the previously identified integral sSJ component, Mesh, in the Malpighian tubule. We genetically manipulated mesh specifically in the principal cells of the tubule at different life stages. Tubules of flies with developmental mesh knockdown revealed defects in epithelial architecture, sSJ molecular and structural organization, and lack of urine production in basal and kinin-stimulated conditions, resulting in edema and early adult lethality. Knockdown of mesh during adulthood did not disrupt tubule epithelial and sSJ integrity but decreased the transepithelial potential, diminished transepithelial fluid and ion transport, and decreased paracellular permeability to 4-kDa dextran. Drosophila kinin decreased transepithelial potential and increased chloride permeability, and it stimulated fluid secretion in both control and adult mesh knockdown tubules but had no effect on 4-kDa dextran flux. Together, these data indicate roles for Mesh in the developmental maturation of the Drosophila Malpighian tubule and in ion and macromolecular transport in the adult tubule.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Proteínas de Drosophila/deficiência , Epitélio/metabolismo , Epitélio/ultraestrutura , Túbulos de Malpighi/metabolismo , Túbulos de Malpighi/ultraestrutura , Proteínas de Membrana/deficiência , Morfogênese/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Transporte de Íons/fisiologia , Proteínas de Membrana/genética
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