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1.
Nat Commun ; 11(1): 5084, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033253

RESUMO

Identifying factors underlying resistance to immune checkpoint therapy (ICT) is still challenging. Most cancer patients do not respond to ICT and the availability of the predictive biomarkers is limited. Here, we re-analyze a publicly available single-cell RNA sequencing (scRNA-seq) dataset of melanoma samples of patients subjected to ICT and identify a subset of macrophages overexpressing TREM2 and a subset of gammadelta T cells that are both overrepresented in the non-responding tumors. In addition, the percentage of a B cell subset is significantly lower in the non-responders. The presence of these immune cell subtypes is corroborated in other publicly available scRNA-seq datasets. The analyses of bulk RNA-seq datasets of the melanoma samples identify and validate a signature - ImmuneCells.Sig - enriched with the genes characteristic of the above immune cell subsets to predict response to immunotherapy. ImmuneCells.Sig could represent a valuable tool for clinical decision making in patients receiving immunotherapy.


Assuntos
Perfilação da Expressão Gênica , Imunoterapia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores Imunológicos/metabolismo , Linfócitos T/metabolismo , Área Sob a Curva , Linfócitos B/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Macrófagos/patologia , Melanoma/genética , Melanoma/patologia , Reprodutibilidade dos Testes
2.
Nat Commun ; 11(1): 4283, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883967

RESUMO

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.


Assuntos
Matriz Extracelular/metabolismo , Fator 4 de Crescimento de Fibroblastos/metabolismo , Coração , Células-Tronco Embrionárias Murinas/metabolismo , Organoides , Potenciais de Ação , Diamino Aminoácidos/metabolismo , Animais , Biomimética/métodos , Diferenciação Celular , Linhagem Celular , Células Endoteliais , Coração/crescimento & desenvolvimento , Coração/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Contração Miocárdica , Miocárdio , Organoides/citologia , Organoides/crescimento & desenvolvimento , Organoides/ultraestrutura
3.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968060

RESUMO

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inativação de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Miosinas/metabolismo , Pseudópodes/metabolismo , Receptor EphB2
4.
N Engl J Med ; 383(12): 1149-1155, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32937047

RESUMO

Daratumumab, a human monoclonal antibody that targets CD38, depletes plasma cells and is approved for the treatment of multiple myeloma. Long-lived plasma cells are implicated in the pathogenesis of systemic lupus erythematosus because they secrete autoantibodies, but they are unresponsive to standard immunosuppression. We describe the use of daratumumab that induced substantial clinical responses in two patients with life-threatening lupus, with the clinical responses sustained by maintenance therapy with belimumab, an antibody to B-cell activating factor. Significant depletion of long-lived plasma cells, reduction of interferon type I activity, and down-regulation of T-cell transcripts associated with chronic inflammation were documented. (Supported by the Deutsche Forschungsgemeinschaft and others.).


Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Anticorpos Monoclonais/uso terapêutico , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Glicoproteínas de Membrana/antagonistas & inibidores , Plasmócitos/efeitos dos fármacos , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Anticorpos Monoclonais Humanizados/uso terapêutico , Creatinina/sangue , Creatinina/urina , Regulação para Baixo , Feminino , Humanos , Interferon Tipo I/antagonistas & inibidores , Quimioterapia de Manutenção , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteinúria , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
5.
PLoS One ; 15(8): e0237577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790741

RESUMO

Abnormal skin melanin homeostasis results in refractory pigmentary diseases. Melanogenesis is influenced by gene regulation, ultraviolet radiation, and host epigenetic responses. Steroid receptor RNA activator (SRA), a long noncoding RNA, is known to regulate steroidogenesis and tumorigenesis. However, how SRA contributes to melanogenesis remains unknown. Using RNA interference against SRA in B16 and A375 melanoma cells, we observed increased pigmentation and increased expression of TRP1 and TRP2 at transcriptional and translational levels only in B16 cells. The constitutive phosphorylation of p38 in B16-shCtrl cells was inhibited in cells with knocked down SRAi. Moreover, the melanin content of control B16 cells was increased by SB202190, a p38 inhibitor. Furthermore, reduced p38 phosphorylation, enhanced TRP1 expression, and hypermelanosis were observed in A375 cells with RNA interference. These results indicate that SRA-p38-TRP1 axis has a regulatory role in melanin homeostasis and that SRA might be a potential therapeutic target for treating pigmentary diseases.


Assuntos
Oxirredutases Intramoleculares/metabolismo , Melaninas/metabolismo , Melanoma Experimental/patologia , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Oxirredutases Intramoleculares/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Oxirredutases/genética , Fosforilação , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
6.
Virology ; 548: 17-24, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838939

RESUMO

The demyelinating disease progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus, JCPyV, under conditions of prolonged immunosuppression. Initial infection is asymptomatic, and the virus establishes lifelong persistence in the host. Following the loss of immune surveillance, the virus can traffic to the central nervous system and infect oligodendrocytes to cause demyelination and PML. The mechanisms involved in glial cell infection are not completely understood. In a screen for N-glycosylated proteins that influence JCPyV pathology, we identified Adipocyte Plasma Membrane Associated Protein (APMAP) as a host cell modulator of JCPyV infection. The removal of APMAP by small interfering siRNA as well as by CRISPR-Cas9 gene editing resulted in a significant decrease in JCPyV infection. Exogenous expression of APMAP in APMAP knockout cell lines rescued susceptibility to infection. These data suggest that virus infection of glial cells is dependent on APMAP.


Assuntos
Vírus JC/fisiologia , Neuroglia/metabolismo , Infecções por Polyomavirus/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Vírus JC/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Neuroglia/virologia , Oligodendroglia/metabolismo , Oligodendroglia/virologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/virologia
7.
Nat Commun ; 11(1): 4017, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782292

RESUMO

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.


Assuntos
Microbioma Gastrointestinal , Hexosaminidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Animais , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hexosaminidases/química , Hexosaminidases/genética , Humanos , Glicoproteínas de Membrana/química , Estrutura Molecular , Mucinas/química , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
8.
Proc Natl Acad Sci U S A ; 117(37): 23054-23065, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32855296

RESUMO

The 100-y-old neuron doctrine from Ramón y Cajal states that neurons are individual cells, rejecting the process of cell-cell fusion in the normal development and function of the nervous system. However, fusogens-specialized molecules essential and sufficient for the fusion of cells-are expressed in the nervous system of different species under conditions of viral infection, stress, or disease. Despite these findings, whether the expression of fusogens in neurons leads to cell-cell fusion, and, if so, whether this affects neuronal fate, function, and animal behavior, has not been explored. Here, using Caenorhabditis elegans chemosensory neurons as a model system, we provide proof-of-principle that aberrant expression of fusogens in neurons results in neuron-neuron fusion and behavioral impairments. We demonstrate that fusion between chemoattractive neurons does not affect the response to odorants, whereas fusion between chemoattractive and chemorepulsive neurons compromises chemosensation. Moreover, we provide evidence that fused neurons are viable and retain their original specific neuronal fate markers. Finally, analysis of calcium transients reveals that fused neurons become electrically coupled, thereby compromising neural circuit connectivity. Thus, we propose that aberrant expression of fusogens in the nervous system disrupts neuronal individuality, which, in turn, leads to a change in neural circuit connectivity and disruption of normal behavior. Our results expose a previously uncharacterized basis of circuit malfunction, and a possible underlying cause of neurological diseases.


Assuntos
Comportamento Animal/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Neurônios/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Comunicação Celular/fisiologia , Fusão Celular/métodos , Glicoproteínas de Membrana/metabolismo , Sistema Nervoso/metabolismo , Neurônios/metabolismo
9.
Arterioscler Thromb Vasc Biol ; 40(9): 2293-2309, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32757648

RESUMO

OBJECTIVE: Extracellular vesicles (EVs) have the potential to act as intercellular communicators. The aims were to characterize circulating EVs in patients with pulmonary arterial hypertension (PAH) and to explore whether these EVs contribute to endothelial activation and angiogenesis. Approach and Results: Patients with PAH (n=70) and healthy controls (HC; n=20) were included in this cross-sectional study. EVs were characterized and human pulmonary endothelial cells (hPAECs) were incubated with purified EVs. Endothelial cell activity and proangiogenic markers were analyzed. Tube formation analysis was performed for hPAECs, and the involvement of PSGL-1 (P-selectin glycoprotein ligand 1) was evaluated. The numbers of CD62P+, CD144+, and CD235a EVs were higher in blood from PAH compared with HC. Thirteen proteins were differently expressed in PAH and HC EVs, where complement fragment C1q was the most significantly elevated protein (P=0.0009) in PAH EVs. Upon EVs-internalization in hPAECs, more PAH compared with HC EVs evaded lysosomes (P<0.01). As oppose to HC, PAH EVs stimulated hPAEC activation and induced transcription and translation of VEGF-A (vascular endothelial growth factor A; P<0.05) and FGF (fibroblast growth factor; P<0.005) which were released in the cell supernatant. These proangiogenic proteins were higher in patient with PAH plasma compered with HC. PAH EVs induced a complex network of angiotubes in vitro, which was abolished by inhibitory PSGL-1antibody. Anti-PSGL-1 also inhibited EV-induced endothelial cell activation and PAH EV dependent increase of VEGF-A. CONCLUSIONS: Patients with PAH have higher levels of EVs harboring increased amounts of angiogenic proteins, which induce activation of hPAECs and in vitro angiogenesis. These effects were partly because of platelet-derived EVs evasion of lysosomes upon internalization within hPAEC and through possible involvement of P-selectin-PSGL-1 pathway.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Neovascularização Fisiológica , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Idoso , Estudos de Casos e Controles , Células Cultivadas , Estudos Transversais , Células Endoteliais/ultraestrutura , Endotélio Vascular/fisiopatologia , Endotélio Vascular/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Selectina-P/metabolismo , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/ultraestrutura , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
10.
PLoS One ; 15(8): e0238466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857809

RESUMO

PURPOSE: Phyllodes tumors (PTs) are biphasic tumors accounting for 0.3-1.5% of all breast tumors. Epithelial membrane proteins (EMPs) have been reported in various malignant tumors but their expression in PTs is unclear. In this study, we aimed to evaluate the expression of EMP1, EMP2, and EMP3 in breast phyllodes tumors (PTs), and to investigate their clinical implications. METHODS: In total, 185 PTs were used for constructing a tissue microarray. Immunohistochemical staining for EMP1, EMP2, and EMP3 was performed, and the results were analyzed along with the clinicopathologic parameters. RESULTS: In total, 185 PTs were included in this study, and comprised 138 benign, 32 borderline, and 15 malignant PTs. In malignant PTs, the epithelial component showed decreased expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032), compared to the benign and borderline PTs. Conversely, stromal component of borderline and malignant PTs showed higher expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032) compared to benign PTs. Expression of EMP1 and EMP3 correlated positively with stromal cellularity and cellular atypia (P < 0.001). In the univariate analysis, stromal EMP3 was associated with shorter disease-free survival (P < 0.001), and shorter overall survival (P = 0.034). CONCLUSION: The expression of EMP1, EMP2, and EMP3 is decreased in the epithelial component and is increased in the stromal component of PT with higher histologic grade. Thus, stromal EMP3 expression may serve as an independent prognostic factor in PT.


Assuntos
Neoplasias da Mama/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Tumor Filoide/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Gradação de Tumores , Tumor Filoide/patologia , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida , Análise Serial de Tecidos
11.
Nat Commun ; 11(1): 3935, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769979

RESUMO

GABAA/glycine-mediated neuronal inhibition critically depends on intracellular chloride (Cl-) concentration which is mainly regulated by the K+-Cl- co-transporter 2 (KCC2) in the adult central nervous system (CNS). KCC2 heterogeneity thus affects information processing across CNS areas. Here, we uncover a gradient in Cl- extrusion capacity across the superficial dorsal horn (SDH) of the spinal cord (laminae I-II: LI-LII), which remains concealed under low Cl- load. Under high Cl- load or heightened synaptic drive, lower Cl- extrusion is unveiled in LI, as expected from the gradient in KCC2 expression found across the SDH. Blocking TrkB receptors increases KCC2 in LI, pointing to differential constitutive TrkB activation across laminae. Higher Cl- lability in LI results in rapidly collapsing inhibition, and a form of activity-dependent synaptic plasticity expressed as a continuous facilitation of excitatory responses. The higher metaplasticity in LI as compared to LII differentially affects sensitization to thermal and mechanical input. Thus, inconspicuous heterogeneity of Cl- extrusion across laminae critically shapes plasticity for selective nociceptive modalities.


Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Cloretos/metabolismo , Plasticidade Neuronal/fisiologia , Nociceptividade/fisiologia , Células do Corno Posterior/fisiologia , Animais , Células Cultivadas , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Neurológicos , Optogenética , Cultura Primária de Células , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptor trkB/antagonistas & inibidores , Receptor trkB/metabolismo , Simportadores/metabolismo
13.
Nat Commun ; 11(1): 3569, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678083

RESUMO

The clinically important MAM blood group antigen is present on haematopoietic cells of all humans except rare MAM-negative individuals. Its molecular basis is unknown. By whole-exome sequencing we identify EMP3, encoding epithelial membrane protein 3 (EMP3), as a candidate gene, then demonstrate inactivating mutations in ten known MAM-negative individuals. We show that EMP3, a purported tumour suppressor in various solid tumours, is expressed in erythroid cells. Disruption of EMP3 by CRISPR/Cas9 gene editing in an immortalised human erythroid cell line (BEL-A2) abolishes MAM expression. We find EMP3 to associate with, and stabilise, CD44 in the plasma membrane. Furthermore, cultured erythroid progenitor cells from MAM-negative individuals show markedly increased proliferation and higher reticulocyte yields, suggesting an important regulatory role for EMP3 in erythropoiesis and control of cell production. Our data establish MAM as a new blood group system and demonstrate an interaction of EMP3 with the cell surface signalling molecule CD44.


Assuntos
Antígenos de Grupos Sanguíneos/genética , Proliferação de Células , Células Eritroides/citologia , Glicoproteínas de Membrana/genética , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Plaquetas/metabolismo , Células Cultivadas , Membrana Eritrocítica/metabolismo , Células Eritroides/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Ligação Proteica , Sequenciamento Completo do Exoma
14.
Mol Cell Biol ; 40(19)2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32719109

RESUMO

Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells-referred to as the accessible cholesterol pool-that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.


Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Células 3T3-L1 , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Lipossomos/metabolismo , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espectrometria de Massa de Íon Secundário/métodos , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
15.
Mol Cell Biol ; 40(18)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32631902

RESUMO

hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding but defective for proteasome interaction. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole-cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Ubiquitina Tiolesterase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Citoplasma/metabolismo , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Proteínas Ubiquitinadas/metabolismo
16.
J Transl Med ; 18(1): 233, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: covidwho-592324

RESUMO

BACKGROUND: Epidemiological, virological and pathogenetic characteristics of SARS-CoV-2 infection are under evaluation. A better understanding of the pathophysiology associated with COVID-19 is crucial to improve treatment modalities and to develop effective prevention strategies. Transcriptomic and proteomic data on the host response against SARS-CoV-2 still have anecdotic character; currently available data from other coronavirus infections are therefore a key source of information. METHODS: We investigated selected molecular aspects of three human coronavirus (HCoV) infections, namely SARS-CoV, MERS-CoV and HCoV-229E, through a network based-approach. A functional analysis of HCoV-host interactome was carried out in order to provide a theoretic host-pathogen interaction model for HCoV infections and in order to translate the results in prediction for SARS-CoV-2 pathogenesis. The 3D model of S-glycoprotein of SARS-CoV-2 was compared to the structure of the corresponding SARS-CoV, HCoV-229E and MERS-CoV S-glycoprotein. SARS-CoV, MERS-CoV, HCoV-229E and the host interactome were inferred through published protein-protein interactions (PPI) as well as gene co-expression, triggered by HCoV S-glycoprotein in host cells. RESULTS: Although the amino acid sequences of the S-glycoprotein were found to be different between the various HCoV, the structures showed high similarity, but the best 3D structural overlap shared by SARS-CoV and SARS-CoV-2, consistent with the shared ACE2 predicted receptor. The host interactome, linked to the S-glycoprotein of SARS-CoV and MERS-CoV, mainly highlighted innate immunity pathway components, such as Toll Like receptors, cytokines and chemokines. CONCLUSIONS: In this paper, we developed a network-based model with the aim to define molecular aspects of pathogenic phenotypes in HCoV infections. The resulting pattern may facilitate the process of structure-guided pharmaceutical and diagnostic research with the prospect to identify potential new biological targets.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Modelos Biológicos , Pneumonia Viral/genética , Pneumonia Viral/virologia , Mapeamento de Interação de Proteínas , Humanos , Glicoproteínas de Membrana/metabolismo , Pandemias , Transdução de Sinais/genética , Proteínas do Envelope Viral
17.
Science ; 368(6495): 1132-1135, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499443

RESUMO

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell-secreted epidermal growth factor-like protein, neural epidermal growth factor-like-like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which Nell2 had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.


Assuntos
Comunicação Celular/fisiologia , Endopeptidases/metabolismo , Epididimo/metabolismo , Fertilidade , Infertilidade Masculina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Proteínas ADAM/metabolismo , Animais , Comunicação Celular/genética , Endopeptidases/genética , Infertilidade Masculina/genética , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo
18.
Nat Commun ; 11(1): 2977, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532995

RESUMO

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Glicoproteínas de Membrana/genética , Oncogenes/genética , Isoformas de RNA/genética , Processamento de RNA , Receptor trkB/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Células Cultivadas , Perfilação da Expressão Gênica , Ontologia Genética , Glioma/metabolismo , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de RNA/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/genética
19.
Am J Pathol ; 190(9): 1833-1842, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32473917

RESUMO

Cholestatic liver injury leads to liver dysfunction. The available evidence suggests that platelets can either promote or reduce liver injury and fibrosis. This study focused on the functions of the C-type lectin-like receptor 2 (CLEC-2), a new special platelet receptor that binds with podoplanin-activating platelets. The role of CLEC-2 and podoplanin in cholestatic liver injury was investigated. Mice were injected intraperitoneally with weekly doses of anti-CLEC-2 antibody (2A2B10) to achieve effective CLEC-2 inhibition in their platelets. Next, left and middle hepatic bile duct ligation (BDL) procedures were performed, and mice were euthanized 1 week later (2A2B10-BDL group). In addition, mice were prepared for control groups, and relevant histological and laboratory variables were compared among these groups. The inhibition of CLEC-2 resulted in increasing hepatocellular necrosis, hepatic inflammation, and liver fibrosis. In addition, podoplanin was strongly expressed in hepatic sinusoidal endothelial cells in BDL-treated mice. Moreover, in 2A2B10-BDL mice, total plasma bile acid levels were significantly increased. In summary, podoplanin is expressed on hepatic sinusoidal endothelial cells upon BDL. Platelets bind with podoplanin via CLEC-2 and become activated. As a result, the total bile acid pool is decreased. Therefore, the CLEC-2-podoplanin interaction promotes liver protection and inhibits liver fibrosis after cholestatic liver injury.


Assuntos
Plaquetas/metabolismo , Colestase/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Colestase/patologia , Células Endoteliais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação Plaquetária/fisiologia
20.
APMIS ; 128(9): 531-538, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32578252

RESUMO

Despite the interest of researchers in IgG4-related disease (IgG4-RD), many questions still remain unanswered regarding the thyroid gland. We aimed to clarify the relationship between IgG4-positive plasma cells and the histopathological pattern in the Hashimoto thyroiditis (HT) in a Finnish series. HT specimens (n = 280) were retrieved from the Department of Pathology, Fimlab Laboratories. After re-evaluation, 82 (29%) cases (72 females and 10 males, 52 ± 17 years) with significant fibrosis were selected. CD38, IgG and IgG4 positivity in plasma cells was evaluated by immunohistochemistry. Adjusted IgG4-positive plasma cells per HPF > 20 and IgG4- to IgG-positive plasma cell ratio > 30% were adopted as threshold criteria and related to other morphological features. IgG4-positive HT group included 13 cases (15% from fibrotic HT, 4.6% from all HT, 50 ± 15 years, 11 females) with adjusted HPF count 30 ± 5 (23-40) IgG4-positive cells. IgG4-positivity significantly correlated with the presence of lobulation, oncocytic metaplasia and certain type of fibrosis, fibrosis spread outside the gland, lymphocytes/plasma cells epithelial penetration, the predominance of microfollicles and follicular atrophy in the present study. Despite the persisting uncertainty whether HT is IgG4-RD, HT with IgG4-positive plasma cells is histopathologically distinct entity with some geographic variability.


Assuntos
Doença de Hashimoto/imunologia , Imunoglobulina G/metabolismo , Plasmócitos/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Fibrose , Finlândia , Doença de Hashimoto/patologia , Humanos , Inflamação , Contagem de Linfócitos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Plasmócitos/patologia , Glândula Tireoide/imunologia , Glândula Tireoide/patologia , Adulto Jovem
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