Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 560
Filtrar
2.
Mol Cell ; 78(4): 779-784.e5, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32362314

RESUMO

The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Furina/química , Furina/genética , Furina/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/virologia , Pandemias , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Ligação Viral
4.
Basic Res Cardiol ; 115(3): 31, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32274570

RESUMO

From January 2020, coronavirus disease (COVID-19) originated in China has spread around the world. The disease is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The presence of myocarditis, cardiac arrest, and acute heart failure in COVID-19 patients suggests the existence of a relationship between SARS-CoV-2 infection and cardiac disease. The Notch signalling is a major regulator of cardiovascular function and it is also implicated in several biological processes mediating viral infections. In this report we discuss the possibility to target Notch signalling to prevent SARS-CoV-2 infection and interfere with the progression of COVID-19- associated heart and lungs disease.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/fisiopatologia , Cardiopatias/tratamento farmacológico , Cardiopatias/etiologia , Pneumopatias/tratamento farmacológico , Pneumopatias/etiologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/fisiopatologia , Receptores Notch/antagonistas & inibidores , Proteína ADAM17/antagonistas & inibidores , Betacoronavirus/efeitos dos fármacos , China , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Progressão da Doença , Furina/metabolismo , Parada Cardíaca/etiologia , Parada Cardíaca/patologia , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Humanos , Interleucina-6/imunologia , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Miocardite/etiologia , Miocardite/patologia , Pandemias , Peptidil Dipeptidase A/deficiência , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Nat Mater ; 18(12): 1376-1383, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31636420

RESUMO

Among the strategies used for enhancement of tumour retention of imaging agents or anticancer drugs is the rational design of probes that undergo a tumour-specific enzymatic reaction preventing them from being pumped out of the cell. Here, the anticancer agent olsalazine (Olsa) was conjugated to the cell-penetrating peptide RVRR. Taking advantage of a biologically compatible condensation reaction, single Olsa-RVRR molecules were self-assembled into large intracellular nanoparticles by the tumour-associated enzyme furin. Both Olsa-RVRR and Olsa nanoparticles were readily detected with chemical exchange saturation transfer magnetic resonance imaging by virtue of exchangeable Olsa hydroxyl protons. In vivo studies using HCT116 and LoVo murine xenografts showed that the OlsaCEST signal and anti-tumour therapeutic effect were 6.5- and 5.2-fold increased, respectively, compared to Olsa without RVRR, with an excellent 'theranostic correlation' (R2 = 0.97) between the imaging signal and therapeutic response (normalized tumour size). This furin-targeted, magnetic resonance imaging-detectable platform has potential for imaging tumour aggressiveness, drug accumulation and therapeutic response.


Assuntos
Ácidos Aminossalicílicos/metabolismo , Antineoplásicos/metabolismo , Furina/metabolismo , Espaço Intracelular/metabolismo , Imagem por Ressonância Magnética/métodos , Nanopartículas , Ácidos Aminossalicílicos/química , Animais , Antineoplásicos/química , Catálise , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células HCT116 , Humanos , Camundongos
8.
Pflugers Arch ; 471(11-12): 1383-1396, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31654198

RESUMO

The epithelial Na+ channel (ENaC) is essential for Na+/K+ homeostasis and blood pressure control. Its activity is regulated by proteases in rodents. To gain more information on proteolytic ENaC regulation in humans, we tested the hypotheses that (1) human kidney α- and γ-ENaC subunits are furin-cleaved, glycosylated, and altered by medication that change plasma aldosterone; (2) prostasin-cleaved γ-ENaC is increased in proteinuria, and (3) cleaved ENaC moieties prevail at the membranes and in urinary extracellular vesicles (uEVs). We developed three monoclonal antibodies (mAbs) targeting (1) the neo-epitope generated after furin cleavage in γ-ENaC (mAb-furin); (2) the intact prostasin cleavage-site in γ-ENaC (mAb-intactRKRK), and (3) the α-ENaC subunit (mAb-alpha). Nephrectomy tissue and uEVs were used for immunoblotting and -histochemistry. In human kidney tissue, mAb-furin detected a ≈ 65-70 kDa protein, compatible with furin-cleaved γ-ENaC; mAb-intactRKRK detected full-length (≈ 90-100 kDa) and furin-cleaved (≈ 70-75 kDa) γ-ENaC. mAb-alpha detected a ≈ 50 kDa protein compatible with furin-cleaved α-subunit. Furin-cleaved γ-ENaC was detected predominantly within membrane fractions and deglycosylation shifted full-length γ-ENaC migration ~ 20 kDa. While γ-ENaC uEV levels were below the detection limit, α-ENaC migrated as intact (≈ 75 kDa) and furin-cleaved (≈ 50 kDa) in uEVs. Kidney levels of α- and γ-ENaC in diuretic- (n = 3) and ACE-inhibitor-treated (n = 4) patients were not different from controls (n = 4). Proteinuric patients (n = 6) displayed similar level of furin-cleaved γ-ENaC as controls (n = 4). Cleaved α-ENaC abundance was significantly lower in the kidneys from proteinuria patients. In conclusion, the study demonstrates ENaC cleavage as an event in human kidney that could contribute to physiological regulation and pathophysiological activation of ENaC.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Epitélio/metabolismo , Furina/metabolismo , Rim/metabolismo , Subunidades Proteicas/metabolismo , Canais de Sódio/metabolismo , Aldosterona/metabolismo , Animais , Diuréticos/farmacologia , Epitélio/efeitos dos fármacos , Glicosilação , Humanos , Rim/efeitos dos fármacos , Camundongos , Proteinúria/metabolismo , Serina Endopeptidases/metabolismo , Sódio/metabolismo
9.
Nat Genet ; 51(10): 1475-1485, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548722

RESUMO

The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.


Assuntos
Regulação da Expressão Gênica , Predisposição Genética para Doença , Células-Tronco Pluripotentes Induzidas/patologia , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Esquizofrenia/genética , Esquizofrenia/patologia , Sistemas CRISPR-Cas , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Feminino , Furina/antagonistas & inibidores , Furina/genética , Furina/metabolismo , Edição de Genes , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Proteínas Monoméricas de Montagem de Clatrina/antagonistas & inibidores , Proteínas Monoméricas de Montagem de Clatrina/genética , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas SNARE/antagonistas & inibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo
10.
J Biol Chem ; 294(37): 13769-13780, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346034

RESUMO

The assembly of collagen VI microfibrils is a multistep process in which proteolytic processing within the C-terminal globular region of the collagen VI α3 chain plays a major role. However, the mechanisms involved remain elusive. Moreover, C5, the short and most C-terminal domain of the α3 chain, recently has been proposed to be released as an adipokine that enhances tumor progression, fibrosis, inflammation, and insulin resistance and has been named "endotrophin." Serum endotrophin could be a useful biomarker to monitor the progression of such disorders as chronic obstructive pulmonary disease, systemic sclerosis, and kidney diseases. Here, using biochemical and isotopic MS-based analyses, we found that the extracellular metalloproteinase bone morphogenetic protein 1 (BMP-1) is involved in endotrophin release and determined the exact BMP-1 cleavage site. Moreover, we provide evidence that several endotrophin-containing fragments are present in various tissues and body fluids. Among these, a large C2-C5 fragment, which contained endotrophin, was released by furin-like proprotein convertase cleavage. By using immunofluorescence microscopy and EM, we also demonstrate that these proteolytic maturations occur after secretion of collagen VI tetramers and during microfibril assembly. Differential localization of N- and C-terminal regions of the collagen VI α3 chain revealed that cleavage products are deposited in tissue and cell cultures. The detailed information on the processing of the collagen VI α3 chain reported here provides a basis for unraveling the function of endotrophin (C5) and larger endotrophin-containing fragments and for refining their use as biomarkers of disease progression.


Assuntos
Proteína Morfogenética Óssea 1/metabolismo , Colágeno Tipo VI/metabolismo , Pró-Proteína Convertases/metabolismo , Fibrose , Furina/metabolismo , Células HEK293 , Humanos , Resistência à Insulina , Microfibrilas/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteólise
11.
Dis Model Mech ; 12(6)2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31101650

RESUMO

Global inhibition of N-linked glycosylation broadly reduces glycan occupancy on glycoproteins, but identifying how this inhibition functionally impacts specific glycoproteins is challenging. This limits our understanding of pathogenesis in the congenital disorders of glycosylation (CDG). We used selective exo-enzymatic labeling of cells deficient in the two catalytic subunits of oligosaccharyltransferase - STT3A and STT3B - to monitor the presence and glycosylation status of cell surface glycoproteins. We show reduced abundance of two canonical tyrosine receptor kinases - the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) - at the cell surface in STT3A-null cells, due to decreased N-linked glycan site occupancy and proteolytic processing in combination with increased endoplasmic reticulum localization. Providing cDNA for Golgi-resident proprotein convertase subtilisin/kexin type 5a (PCSK5a) and furin cDNA to wild-type and mutant cells produced under-glycosylated forms of PCSK5a, but not furin, in cells lacking STT3A. Reduced glycosylation of PCSK5a in STT3A-null cells or cells treated with the oligosaccharyltransferase inhibitor NGI-1 corresponded with failure to rescue receptor processing, implying that alterations in the glycosylation of this convertase have functional consequences. Collectively, our findings show that STT3A-dependent inhibition of N-linked glycosylation on receptor tyrosine kinases and their convertases combines to impair receptor processing and surface localization. These results provide new insight into CDG pathogenesis and highlight how the surface abundance of some glycoproteins can be dually impacted by abnormal glycosylation.


Assuntos
Processamento de Proteína Pós-Traducional , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Furina/metabolismo , Glicosilação , Células HEK293 , Hexosiltransferases/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Pró-Proteína Convertase 5/metabolismo , Proteômica , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo
12.
PLoS One ; 14(3): e0213438, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30861020

RESUMO

A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.


Assuntos
Furina/biossíntese , Furina/genética , Tabaco/genética , Tabaco/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Fator IX/genética , Fator IX/metabolismo , Furina/metabolismo , Humanos , Técnicas In Vitro , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Camundongos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Engenharia de Proteínas/métodos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo
13.
PLoS One ; 14(3): e0212992, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840660

RESUMO

The homeodomain transcription factor NKX2-5 is known to be essential for both normal heart development and for heart function. But little is yet known about the identities of its downstream effectors or their function during differentiation of cardiac progenitor cells (CPCs). We have used transgenic analysis and CRISPR-mediated ablation to identify a cardiac enhancer of the Furin gene. The Furin gene, encoding a proprotein convertase, is directly repressed by NKX2-5. Deletion of Furin in CPCs is embryonic lethal, with mutant hearts showing a range of abnormalities in the outflow tract. Those defects are associated with a reduction in proliferation and premature differentiation of the CPCs. Deletion of Furin in differentiated cardiomyocytes results in viable adult mutant mice showing an elongation of the PR interval, a phenotype that is consistent with the phenotype of mice and human mutant for Nkx2-5. Our results show that Furin mediate some aspects of Nkx2-5 function in the heart.


Assuntos
Furina/genética , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteína Homeobox Nkx-2.5/metabolismo , Organogênese/genética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Proliferação de Células/genética , Embrião de Mamíferos , Furina/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Mutagênese , Mutação , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Células-Tronco/fisiologia
14.
ACS Appl Mater Interfaces ; 11(13): 12327-12334, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30864434

RESUMO

Self-assembled nanostructures of amphiphilic peptides have a wide range of applications in bioimaging and delivery systems. In this study, we design and synthesize a biocompatible amphiphilic peptide (C-3) consisting of an RVRRFFF sequence and a nitrobenzoxadiazole fluorophore that can self-assemble into stable micelles for specifically detecting furin, a kind of proprotein convertase with promoting tumor progression. The self-assembly of C-3 with a ß-sheet nanostructure is capable of a rapid and specific response to furin in only 5 min in aqueous solution because of the existence of the RVRR motif in the C-3 molecule. The C-3 nanostructures thus can selectively distinguish high furin-expressing cancer cells, like MDA-MB-231 cells, a kind of human breast cancer cells, from normal cells. Furthermore, the C-3 self-assembly can stay in living cells for a long time and are capable of durable detection of intracellular furin, being good for tracer analysis. To our knowledge, this is the first example of self-assembly of a soluble amphiphilic peptide that can selectively detect furin in high furin-expressing live cells.


Assuntos
Neoplasias da Mama , Sistemas de Liberação de Medicamentos/métodos , Furina/antagonistas & inibidores , Nanoestruturas/química , Proteínas de Neoplasias/antagonistas & inibidores , Peptídeos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Furina/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Células RAW 264.7
15.
J Cell Biol ; 218(5): 1653-1669, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30808704

RESUMO

How morphogenetic signals are prepared for intercellular dispersal and signaling is fundamental to the understanding of tissue morphogenesis. We discovered an intracellular mechanism that prepares Drosophila melanogaster FGF Branchless (Bnl) for cytoneme-mediated intercellular dispersal during the development of the larval Air-Sac-Primordium (ASP). Wing-disc cells express Bnl as a proprotein that is cleaved by Furin1 in the Golgi. Truncated Bnl sorts asymmetrically to the basal surface, where it is received by cytonemes that extend from the recipient ASP cells. Uncleavable mutant Bnl has signaling activity but is mistargeted to the apical side, reducing its bioavailability. Since Bnl signaling levels feedback control cytoneme production in the ASP, the reduced availability of mutant Bnl on the source basal surface decreases ASP cytoneme numbers, leading to a reduced range of signal/signaling gradient and impaired ASP growth. Thus, enzymatic cleavage ensures polarized intracellular sorting and availability of Bnl to its signaling site, thereby determining its tissue-specific intercellular dispersal and signaling range.


Assuntos
Sacos Aéreos/metabolismo , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Discos Imaginais/metabolismo , Asas de Animais/metabolismo , Sacos Aéreos/citologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Movimento Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Fatores de Crescimento de Fibroblastos/genética , Furina/genética , Furina/metabolismo , Discos Imaginais/citologia , Transporte Proteico , Asas de Animais/citologia
16.
Chem Commun (Camb) ; 55(15): 2218-2221, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30706069

RESUMO

A semisynthetic fluorescent protein assembly-based FRET probe (sFPAP) was proposed for cell membrane protease function assay. Here, the probe was anchored on a living cell membrane through a membrane inserting peptide (MIP) and was successfully utilized for in situ imaging of furin activity on the living cell membrane in real time.


Assuntos
Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Peptídeo Hidrolases/metabolismo , Linhagem Celular Tumoral , Furina/química , Furina/metabolismo , Humanos , Microscopia Confocal , Peptídeo Hidrolases/química , Imagem com Lapso de Tempo
17.
Cell Signal ; 55: 109-118, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633987

RESUMO

Bone morphogenetic protein 6 (BMP6) and transforming growth factor-ß1 (TGF-ß1) are key intraovarian regulators that play essential roles in regulating mammalian follicular function and promoting oocyte maturation. Furin, a member of the subtilisin-like proprotein convertase family, promotes the activation of diverse functional proteins by cleaving protein precursors in the secretory pathway. The aim of this study was to investigate the effect and underlying molecular mechanisms by which BMP6 regulates the expression of furin to increase TGF-ß1 production. Primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used as study models. Our results show that BMP6 significantly up-regulated the expression of furin and increased the production of TGF-ß1 in hGL cells. Using dual inhibition approaches (kinase receptor inhibitors and small interfering RNA-targeted knockdown), we demonstrate that both activin receptor-like (ALK)2 and ALK3 are involved in the BMP6-induced up-regulation of furin. Additionally, knockdown of furin abolished BMP6-induced increases in TGF-ß1 production. Moreover, knockdown of endogenous SMAD4 reversed the BMP6-induced increase in furin expression. These results indicate that the ALK2/3-mediated canonical SMAD signaling pathway is required for the stimulatory effect of BMP6 on furin expression, which in turn increases the production of TGF-ß1 in hGL cells. Our findings provide insights into the molecular interactions and mechanisms of two intrafollicular growth factors in hGL cells.


Assuntos
Proteína Morfogenética Óssea 6/fisiologia , Furina/metabolismo , Células da Granulosa/metabolismo , Células Lúteas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Células Cultivadas , Feminino , Células da Granulosa/citologia , Humanos , Células Lúteas/citologia , Proteína Smad4/metabolismo
18.
Biochim Biophys Acta Mol Basis Dis ; 1865(3): 648-660, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625383

RESUMO

AGel amyloidosis, formerly known as familial amyloidosis of the Finnish-type, is caused by pathological aggregation of proteolytic fragments of plasma gelsolin. So far, four mutations in the gelsolin gene have been reported as responsible for the disease. Although D187N is the first identified variant and the best characterized, its structure has been hitherto elusive. Exploiting a recently-developed nanobody targeting gelsolin, we were able to stabilize the G2 domain of the D187N protein and obtained, for the first time, its high-resolution crystal structure. In the nanobody-stabilized conformation, the main effect of the D187N substitution is the impairment of the calcium binding capability, leading to a destabilization of the C-terminal tail of G2. However, molecular dynamics simulations show that in the absence of the nanobody, D187N-mutated G2 further misfolds, ultimately exposing its hydrophobic core and the furin cleavage site. The nanobody's protective effect is based on the enhancement of the thermodynamic stability of different G2 mutants (D187N, G167R and N184K). In particular, the nanobody reduces the flexibility of dynamic stretches, and most notably decreases the conformational entropy of the C-terminal tail, otherwise stabilized by the presence of the Ca2+ ion. A Caenorhabditis elegans-based assay was also applied to quantify the proteotoxic potential of the mutants and determine whether nanobody stabilization translates into a biologically relevant effect. Successful protection from G2 toxicity in vivo points to the use of C. elegans as a tool for investigating the mechanisms underlying AGel amyloidosis and rapidly screen new therapeutics.


Assuntos
Amiloide/toxicidade , Amiloidose/genética , Distrofias Hereditárias da Córnea/genética , Gelsolina/química , Gelsolina/genética , Gelsolina/metabolismo , Anticorpos de Domínio Único/metabolismo , Substituição de Aminoácidos/genética , Amiloide/genética , Amiloide/metabolismo , Amiloidose/metabolismo , Amiloidose Familiar/genética , Amiloidose Familiar/metabolismo , Animais , Caenorhabditis elegans , Cálcio/química , Cálcio/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Cristalografia por Raios X , Finlândia , Furina/química , Furina/metabolismo , Gelsolina/toxicidade , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidade , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia
19.
Proc Natl Acad Sci U S A ; 116(4): 1279-1288, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30610172

RESUMO

The protein acyl transferase ZDHHC5 was recently proposed to regulate trafficking in the endocytic pathway. Therefore, we explored the function of this enzyme in controlling the action of bacterial toxins. We found that ZDHHC5 activity is required for two very different toxins: the anthrax lethal toxin and the pore-forming toxin aerolysin. Both of these toxins have precursor forms, the protoxins, which can use the proprotein convertases Furin and PC7 for activation. We show that ZDHHC5 indeed affects the processing of the protoxins to their active forms. We found that Furin and PC7 can both be S-palmitoylated and are substrates of ZDHHC5. The impact of ZDHHC5 on Furin/PC7-mediated anthrax toxin cleavage is dual, having an indirect and a direct component. First, ZDHHC5 affects the homeostasis and trafficking of a subset of cellular proteins, including Furin and PC7, presumably by affecting the endocytic/recycling pathway. Second, while not inhibiting the protease activity per se, ZDHHC5-mediated Furin/PC7 palmitoylation is required for the cleavage of the anthrax toxin. Finally, we show that palmitoylation of Furin and PC7 promotes their association with plasma membrane microdomains. Both the receptor-bound toxin and the convertases are of very low abundance at the cell surface. Their encounter is unlikely on reasonable time scales. This work indicates that palmitoylation drives their encounter in specific domains, allowing processing and thereby intoxication of the cell.


Assuntos
Acetiltransferases/metabolismo , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Transporte Proteico/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose/fisiologia , Furina/metabolismo , Células HeLa , Humanos , Microdomínios da Membrana/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Pró-Proteína Convertases/metabolismo , Subtilisinas/metabolismo
20.
Chembiochem ; 20(4): 561-567, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30304583

RESUMO

Live-cell imaging of cell-surface-associated proteolytic enzymes is crucial to understand their biological roles and functions in both physiological and pathological processes. However, the complexity of the cell membrane environment increases difficulties in specifically investigating targeted proteolytic activities within the microenvironment. Towards this end, a unique, photoremovable, furin-responsive peptide probe that can undergo spatiotemporal control through UV-light illumination has been designed and synthesized to aid in visualizing the activity of a cell-surface-associated protease enzyme, furin, in live cells. Prior to light irradiation, the photolabile moiety, 4,5-dimethoxy-2-nitrobenzyl, in the peptide sequence of the reporter will block furin-like enzymatic hydrolysis, and thus, no fluorescence will be observed. Upon simple light illumination, photolysis will occur, thereby revealing the uncaged peptide probe, which can undergo enzyme hydrolysis and lead to an increase in fluorescence signal; this allows the real-time imaging of endogenous cell-surface-associated furin-like enzyme function in living cells through precise spatial and temporal resolution.


Assuntos
Membrana Celular/metabolismo , Furina/metabolismo , Microscopia de Fluorescência , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Hidrólise , Nitrobenzenos/química , Peptídeos/química , Peptídeos/metabolismo , Raios Ultravioleta
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA