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1.
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
2.
Science ; 369(6500): 202-207, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32647003

RESUMO

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.


Assuntos
Actinas/metabolismo , Citocinas/biossíntese , Síndromes de Imunodeficiência/genética , Transtornos Linfoproliferativos/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Membrana/fisiologia , Fator 1 de Ribosilação do ADP/metabolismo , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Humanos , Síndromes de Imunodeficiência/imunologia , Transtornos Linfoproliferativos/imunologia , Proteínas de Membrana/genética , Linhagem , Fosforilação , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
3.
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
4.
Gene ; 753: 144805, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32445923

RESUMO

Genomic variants in both ADTRP and TFPI genes are associated with risk of coronary artery disease (CAD). ADTRP regulates TFPI expression and endothelial cell functions involved in the initiation of atherosclerotic CAD. ADTRP also specifies primitive myelopoiesis and definitive hematopoiesis by upregulating TFPI expression. However, the underlying molecular mechanism is unknown. Here we show that transcription factor POU1F1 is the key by which ADTRP regulates TFPI expression. Luciferase reporter assays, chromatin-immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) in combination with analysis of large and small deletions of the TFPI promoter/regulatory region were used to identify the molecular mechanism by which ADTRP regulates TFPI expression. Genetic association was assessed using case-control association analysis and phenome-wide association analysis (PhenGWA). ADTRP regulates TFPI expression at the transcription level in a dose-dependent manner. The ADTRP-response element was localized to a 50 bp region between -806 bp and -756 bp upstream of TFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI. ChIP and EMSA demonstrated that POU1F1 binds to the ADTRP response element. Genetic analysis identified significant association between POU1F1 variants and risk of CAD. PhenGWA identified other phenotypic traits associated with the ADTRP-POU1F1-TFPI axis such as lymphocyte count (ADTRP), waist circumference (TFPI), and standing height (POU1F1). These data identify POU1F1 as a transcription factor that regulates TFPI transcription in response to ADTRP, and link POU1F1 variants to risk of CAD for the first time.


Assuntos
Doença da Artéria Coronariana/metabolismo , Lipoproteínas/biossíntese , Proteínas de Membrana/metabolismo , Fator de Transcrição Pit-1/metabolismo , Aterosclerose/genética , Estudos de Casos e Controles , Linhagem Celular , Imunoprecipitação da Cromatina/métodos , Doença da Artéria Coronariana/genética , Bases de Dados Genéticas , Células Endoteliais/metabolismo , Genes Homeobox , Células HeLa , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Regiões Promotoras Genéticas , Elementos de Resposta , Sítio de Iniciação de Transcrição , Transcrição Genética
5.
J Pharmacol Sci ; 143(3): 176-181, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32386905

RESUMO

The volume-regulated anion channel (VRAC) plays a central role in maintaining cell volume in response to osmotic stress. Leucine-rich repeat-containing 8A (LRRC8A) was recently identified as an essential component of VRAC although other Cl- channels were also suggested to contribute to VRAC. VRAC is activated when a cell is challenged with a hypotonic environment or even in isotonic conditions challenged with different stimuli. It is not clear how VRAC is activated and whether activation of VRAC in hypotonic and isotonic conditions share the same mechanism. In this present study, we investigated relative contribution of LRRC8A and anoctamin 1(ANO1) to VRAC currents activated by fetal bovine serum (FBS) in isotonic condition, and studied the role of intracellular Ca2+ in this activation. We used CRISPR/Cas9 gene editing approach, electrophysiology, and pharmacology approaches to show that VRAC currents induced by FBS is mostly mediated by LRRC8A in HEK293 cells, but also with significant contribution from ANO1. FBS induces Ca2+ transients and these Ca2+ signals are required for the activation of VRAC by serum. These findings will help to further understand the mechanism in activation of VRAC.


Assuntos
Anoctamina-1/fisiologia , Cálcio/metabolismo , Tamanho Celular , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Canais de Ânion Dependentes de Voltagem/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Animais , Proteína 9 Associada à CRISPR/genética , Bovinos , Canais de Cloreto/metabolismo , Canais de Cloreto/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Células HEK293 , Humanos , Pressão Osmótica/fisiologia , Soro
6.
Proc Natl Acad Sci U S A ; 117(21): 11531-11540, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32414916

RESUMO

A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. Here, we demonstrate dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using the Drosophila follicle epithelium. Our data support a linear hierarchy for localization, but rule out previously proposed protein-protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. Our data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg.


Assuntos
Polaridade Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila , Células Epiteliais , Proteínas de Membrana/fisiologia , Animais , Drosophila/citologia , Drosophila/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Epitélio/fisiologia , Feminino , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Proteína Quinase C , Proteínas Supressoras de Tumor
7.
Acta Trop ; 207: 105464, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32302689

RESUMO

Toxoplasma gondii is an obligatory intracellular parasite that critically depends on active invasion and egress from infected host cells to complete its propagation cycle. T. gondii rhoptry proteins (TgROPs) are virulent factors associated with host cell invasion, growth. In this study, we analyzed the functions of ROP9 in the process of T. gondii infection. The TgROP9 knockout RH strain (RH△ROP9) and its recovery strain (RH-ReROP9) were constructed using the CRISPR/Cas9 system. The invasion, proliferation, and egress efficiency of the RH△ROP9 strain were evaluated and their pathogenicity to mice was analyzed. Compared with RH wild-type (RH-WT) and RH-ReROP9 strains, the invasion percentage of RH△ROP9 to Vero cells was reduced by about 28.0% (p< 0.01) at 1.5 h, and the relative proliferation percentage was decreased by about 35.0% (p< 0.01) after infection with 102 or 103 parasites. In addition, the RH△ROP9 strain also showed prolonged egress time from host cells. The survival time of the mice (12.6 ± 1.6 or 10.1 ± 1.1 days) were delayed (p < 0.001) after infection with either 200 or 1000 RH△ROP9 parasites. These evidences suggested that ROP9 facilitated T. gondii infection in vitro and in vivo.


Assuntos
Antígenos de Protozoários/fisiologia , Proteínas de Membrana/fisiologia , Toxoplasma/patogenicidade , Animais , Chlorocebus aethiops , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Células Vero
8.
PLoS One ; 15(3): e0229834, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155188

RESUMO

MPV17 is described as a mitochondrial inner membrane channel. Although its function remains elusive, mutations in the MPV17 gene result in hepato-cerebral mitochondrial DNA depletion syndrome in humans. In this study, we show that MPV17 silencing does not induce depletion in mitochondrial DNA content in cancer cells. We also show that MPV17 does not control cancer cell proliferation despite the fact that we initially observed a reduced proliferation rate in five MPV17-silenced cancer cell lines with two different shRNAs. However, shRNA-mediated MPV17 knockdown performed in this work provided misguiding results regarding the resulting proliferation phenotype and only a rescue experiment was able to shed definitive light on the implication of MPV17 in cancer cell proliferation. Our results therefore emphasize the caution that is required when scientific conclusions are drawn from a work based on lentiviral vector-based gene silencing and clearly demonstrate the need to systematically perform a rescue experiment in order to ascertain the specific nature of the experimental results.


Assuntos
Proteínas de Membrana/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Neoplasias/patologia , Proliferação de Células , DNA Mitocondrial/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética
9.
Nat Commun ; 11(1): 770, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034138

RESUMO

Invading microbial pathogens can be eliminated selectively by xenophagy. Ubiquitin-mediated autophagy receptors are phosphorylated by TANK-binding kinase 1 (TBK1) and recruited to ubiquitinated bacteria to facilitate autophagosome formation during xenophagy, but the molecular mechanism underlying TBK1 activation in response to microbial infection is not clear. Here, we show that bacterial infection increases Ca2+ levels to activate TBK1 for xenophagy via the Ca2+-binding protein TBC1 domain family member 9 (TBC1D9). Mechanistically, the ubiquitin-binding region (UBR) and Ca2+-binding motif of TBC1D9 mediate its binding with ubiquitin-positive bacteria, and TBC1D9 knockout suppresses TBK1 activation and subsequent recruitment of the ULK1 complex. Treatment with a Ca2+ chelator impairs TBC1D9-ubiquitin interactions and TBK1 activation during xenophagy. TBC1D9 is also recruited to damaged mitochondria through its UBR and Ca2+-binding motif, and is required for TBK1 activation during mitophagy. These results indicate that TBC1D9 controls TBK1 activation during xenophagy and mitophagy through Ca2+-dependent ubiquitin-recognition.


Assuntos
Autofagia/fisiologia , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Infecções Estreptocócicas/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Citosol/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Macroautofagia/fisiologia , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Streptococcus pyogenes/patogenicidade , Ubiquitina/metabolismo
10.
Development ; 147(4)2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32001438

RESUMO

Despites several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, we provide evidence that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so-far-ignored AMP role involves their filopodia-based interactions with nerve growth cones. In parallel, we report the previously undetected expression of the guidance molecule-encoding genes sidestep and side IV in AMPs. Altogether, our data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is crucial for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent, as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs.


Assuntos
Axônios/fisiologia , Neurônios Motores/fisiologia , Músculos/embriologia , Músculos/inervação , Mioblastos/fisiologia , Animais , Apoptose , Orientação de Axônios , Movimento Celular , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Genótipo , Proteínas de Fluorescência Verde , Cones de Crescimento/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Membrana/fisiologia , Microscopia de Fluorescência , Pseudópodes/fisiologia , Transdução de Sinais , Células-Tronco/citologia
11.
Lab Invest ; 100(6): 849-862, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32060407

RESUMO

Wolfram Syndrome 1 (WFS1) protein is an endoplasmic reticulum (ER) factor whose deficiency results in juvenile-onset diabetes secondary to cellular dysfunction and apoptosis. The mechanisms guiding ß-cell outcomes secondary to WFS1 function, however, remain unclear. Here, we show that WFS1 preserves normal ß-cell physiology by promoting insulin biosynthesis and negatively regulating ER stress. Depletion of Wfs1 in vivo and in vitro causes functional defects in glucose-stimulated insulin secretion and insulin content, triggering Chop-mediated apoptotic pathways. Genetic proof of concept studies coupled with RNA-seq reveal that increasing WFS1 confers a functional and a survival advantage to ß-cells under ER stress by increasing insulin gene expression and downregulating the Chop-Trib3 axis, thereby activating Akt pathways. Remarkably, WFS1 and INS levels are reduced in type-2 diabetic (T2DM) islets, suggesting that WFS1 may contribute to T2DM ß-cell pathology. Taken together, this work reveals essential pathways regulated by WFS1 to control ß-cell survival and function primarily through preservation of ER homeostasis.


Assuntos
Células Secretoras de Insulina , Proteínas de Membrana , Animais , Glicemia/análise , Glicemia/metabolismo , Linhagem Celular , Células Cultivadas , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Insulina/análise , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos Knockout , Transdução de Sinais/fisiologia , Síndrome de Wolfram
12.
Am J Respir Cell Mol Biol ; 62(6): 783-792, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32078788

RESUMO

Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-ß mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-ß inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.


Assuntos
Células Epiteliais/virologia , Proteínas de Membrana/fisiologia , Rhinovirus/fisiologia , Replicação Viral , Células A549 , Asma/etiologia , Brônquios/citologia , Células Cultivadas , Cromossomos Humanos Par 17/genética , Estresse do Retículo Endoplasmático , Ácidos Graxos Monoinsaturados/farmacologia , Predisposição Genética para Doença , Genótipo , Células HeLa , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Mucosa Nasal/citologia , Infecções por Picornaviridae/complicações , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Rhinovirus/genética , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Replicação Viral/efeitos dos fármacos
13.
Nat Cell Biol ; 22(3): 289-296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32094692

RESUMO

The process of metastasis is complex1. In breast cancer, there are frequently long time intervals between cells leaving the primary tumour and growth of overt metastases2,3. Reasons for disease indolence and subsequent transition back to aggressive growth include interactions with myeloid and fibroblastic cells in the tumour microenvironment and ongoing immune surveillance4-6. However, the signals that cause actively growing cells to enter an indolent state, thereby enabling them to survive for extended periods of time, are not well understood. Here we reveal how the behaviour of indolent breast cancer cells in the lung is determined by their interactions with alveolar epithelial cells, in particular alveolar type 1 cells. This promotes the formation of fibronectin fibrils by indolent cells that drive integrin-dependent pro-survival signals. Combined in vivo RNA sequencing and drop-out screening identified secreted frizzled-related protein 2 (SFRP2) as a key mediator of this interaction. Sfrp2 is induced in breast cancer cells by signals from lung epithelial cells and promotes fibronectin fibril formation and survival, whereas blockade of Sfrp2 expression reduces the burden of indolent disease.


Assuntos
Células Epiteliais Alveolares/fisiologia , Neoplasias da Mama/patologia , Proteínas de Membrana/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Transdução de Sinais
14.
Science ; 367(6475)2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31949051

RESUMO

Negative checkpoint regulators (NCRs) temper the T cell immune response to self-antigens and limit the development of autoimmunity. Unlike all other NCRs that are expressed on activated T lymphocytes, V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is expressed on naïve T cells. We report an unexpected heterogeneity within the naïve T cell compartment in mice, where loss of VISTA disrupted the major quiescent naïve T cell subset and enhanced self-reactivity. Agonistic VISTA engagement increased T cell tolerance by promoting antigen-induced peripheral T cell deletion. Although a critical player in naïve T cell homeostasis, the ability of VISTA to restrain naïve T cell responses was lost under inflammatory conditions. VISTA is therefore a distinctive NCR of naïve T cells that is critical for steady-state maintenance of quiescence and peripheral tolerance.


Assuntos
Antígenos B7/fisiologia , Proteínas de Membrana/fisiologia , Tolerância Periférica/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Antígenos B7/genética , Ativação Linfocitária , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tolerância Periférica/genética , Receptores de Antígenos de Linfócitos T/fisiologia
15.
Neuron ; 105(6): 1007-1017.e5, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31974009

RESUMO

LRRTM4 is a transsynaptic adhesion protein regulating glutamatergic synapse assembly on dendrites of central neurons. In the mouse retina, we find that LRRTM4 is enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs). Knockout of LRRTM4 reduces RBC axonal GABAA and GABAC receptor clustering and disrupts presynaptic inhibition onto RBC terminals. LRRTM4 removal also perturbs the stereotyped output synapse arrangement at RBC terminals. Synaptic ribbons are normally apposed to two distinct postsynaptic "dyad" partners, but in the absence of LRRTM4, "monad" and "triad" arrangements are also formed. RBCs from retinas deficient in GABA release also demonstrate dyad mis-arrangements but maintain LRRTM4 expression, suggesting that defects in dyad organization in the LRRTM4 knockout could originate from reduced GABA receptor function. LRRTM4 is thus a key synapse organizing molecule at RBC terminals, where it regulates function of GABAergic synapses and assembly of RBC synaptic dyads.


Assuntos
Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Inibição Neural/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Bipolares da Retina/fisiologia , Animais , Feminino , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Receptores de GABA/metabolismo , Receptores de GABA/fisiologia , Retina/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Ácido gama-Aminobutírico/metabolismo
16.
Vet Microbiol ; 241: 108555, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31928702

RESUMO

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.


Assuntos
Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/análise , Macrófagos/microbiologia , Proteínas de Membrana/fisiologia , Animais , Galinhas , Biologia Computacional , Meios de Cultura/química , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/fisiologia , Deleção de Genes , Expressão Gênica , Concentração de Íons de Hidrogênio , Análise em Microsséries/veterinária , Mutação , Nitrogênio/deficiência , Doenças das Aves Domésticas/microbiologia , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , RNA Complementar/química , RNA Complementar/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Organismos Livres de Patógenos Específicos , Virulência , beta-Galactosidase/metabolismo
17.
J Cell Biol ; 219(2)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31985747

RESUMO

IRE1ß is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1ß have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1ß diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1ß can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1ß has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1ß to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host-environment interface.


Assuntos
Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Células CACO-2 , Endorribonucleases/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas Serina-Treonina Quinases/genética , Proteostase , Análise de Sequência de Proteína , Transdução de Sinais , Estresse Fisiológico , Resposta a Proteínas não Dobradas
18.
Mol Immunol ; 119: 8-17, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31927202

RESUMO

Motile sperm domain containing 2 (MOSPD2) is a single-pass membrane protein to which until recently little function had been ascribed. Although its mammalian homologs have been identified, the status of the mospd2 gene in lower vertebrates is still unknown. In the present study, cDNA of the mospd2 gene of barbel steed (Hemibarbus labeo) was cloned and sequenced to characterize its potential involvement in the innate immune system of this fish. Sequence analysis revealed that the predicted barbel steed MOSPD2 protein contained an N-terminal extracellular portion composed of a CRAL-TRIO domain, a motile sperm domain, and a transmembrane domain, as well as a short C-terminal intracellular domain. Phylogenetic tree analysis indicated that barbel steed MOSPD2 is closely related to that of zebrafish. Barbel steed mospd2 transcripts were detected in a wide range of tissues, with the highest level being found in the gill. In response to lipopolysaccharide (LPS) treatment or Aeromonas hydrophila infection, mospd2 gene expression was significantly altered in the head kidney, spleen, and mid-intestine. The expression of mospd2 gene was detected in monocytes/macrophages (MO/MФ), neutrophils, and lymphocytes, and was found to be mainly expressed in MO/MФ. At the same time, using flow cytometry, we also confirmed that MOSPD2 protein is located on MO/MФ, neutrophil, and lymphocyte membranes. Following treatment with LPS or A. hydrophila, MOSPD2 protein expression was induced in these immune cells. The migration of MO/MФ and neutrophils decreased significantly upon MOSPD2 blockade with anti-MOSPD2 IgG in a dose-dependent manner, whereas this treatment had no significant effect on lymphocytes migration. To the best of our knowledge, our study, for the first time, provides evidence that MOSPD2 mediates the migration of MO/MФ and neutrophils in a fish species.


Assuntos
Quimiotaxia/fisiologia , Cyprinidae/fisiologia , Proteínas de Peixes/fisiologia , Proteínas de Membrana/fisiologia , Aeromonas hydrophila/imunologia , Animais , Clonagem Molecular , Cyprinidae/genética , Cyprinidae/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Macrófagos/fisiologia , Proteínas de Membrana/genética , Monócitos/fisiologia , Neutrófilos/fisiologia , Análise de Sequência de DNA
19.
Mol Cell ; 77(3): 618-632.e5, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31806350

RESUMO

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1 from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.


Assuntos
Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Transporte Proteico/fisiologia
20.
Neuron ; 105(2): 310-321.e3, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31761710

RESUMO

Transmembrane channel-like (TMC) 1 and 2 are required for the mechanotransduction of mouse inner ear hair cells and localize to the site of mechanotransduction in mouse hair cell stereocilia. However, it remains unclear whether TMC1 and TMC2 are indeed ion channels and whether they can sense mechanical force directly. Here we express TMC1 from the green sea turtle (CmTMC1) and TMC2 from the budgerigar (MuTMC2) in insect cells, purify and reconstitute the proteins, and show that liposome-reconstituted CmTMC1 and MuTMC2 proteins possess ion channel activity. Furthermore, by applying pressure to proteoliposomes, we demonstrate that both CmTMC1 and MuTMC2 proteins can indeed respond to mechanical stimuli. In addition, CmTMC1 mutants corresponding to human hearing loss mutants exhibit reduced or no ion channel activity. Taken together, our results show that the CmTMC1 and MuTMC2 proteins are pore-forming subunits of mechanosensitive ion channels, supporting TMC1 and TMC2 as hair cell transduction channels.


Assuntos
Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/fisiologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Feminino , Lipossomos/metabolismo , Melopsittacus , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Mutação , Spodoptera , Tartarugas
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