RESUMO
In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.
Assuntos
Betacoronavirus/química , Furina/química , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Genoma Viral , Conformação Proteica , SARS-CoV-2RESUMO
We have previously demonstrated that measurement of tissue concentrations of the secretogranin II (SgII or SCG2 as listed in the HUGO database)-derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas and that EM66 represents a sensitive plasma marker of pheochromocytomas. Here, we investigated the gene expression and protein production of SgII in 13 normal adrenal glands, and 35 benign and 16 malignant pheochromocytomas with the goal to examine the molecular mechanisms leading to the marked variations in the expression of EM66 in tumoral chromaffin tissue. EM66 peptide levels were 16-fold higher in benign than in malignant pheochromocytomas and had an area under the receiver-operating characteristic curve of 0.95 for the distinction of benign and malignant tumors. Q-PCR experiments indicated that the SgII gene was significantly underexpressed in malignant tumors compared with benign tumors. Western blot analysis using antisera directed against SgII and SgII-derived fragments revealed lower SgII protein and SgII-processing products in malignant tumors. Western blot also showed that low p-cAMP-responsive element-binding (CREB) concentrations seemed to be associated with the malignant status. In addition, the prohormone convertase PC1 and PC2 genes and proteins were overexpressed in benign pheochromocytomas compared with malignant pheochromocytomas. Low concentrations of EM66 found in malignant tumors are associated with reduced expression and production of SgII and SgII-derived peptides that could be ascribed to a decrease in SgII gene transcription, probably linked to p-CREB down-regulation, and to lower PC levels. These findings highlight the mechanisms leading to lower concentrations of EM66 in malignant pheochromocytoma and strengthen the notion that this peptide is a suitable marker of this neuroendocrine tumor.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Biomarcadores Tumorais/metabolismo , Fragmentos de Peptídeos/metabolismo , Feocromocitoma/metabolismo , Secretogranina II/metabolismo , Adolescente , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/fisiologia , Adulto , Idoso , Biomarcadores Tumorais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Feocromocitoma/genética , Feocromocitoma/patologia , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/genética , Pró-Proteína Convertase 2/metabolismo , Secretogranina II/genética , Adulto JovemRESUMO
BACKGROUND AND AIMS: Hepcidin is an iron homoeostasis regulator peptide. Loss-of-function mutations cause juvenile haemochromatosis while its over-expression results in anaemia. However, the mechanism and function of preprohepcidin conversion to mature hepcidins (25, 22 and 20 amino acid C-terminal peptides) are not well known. After removal of the signal peptide, the first proteolytic cleavage occurs within the basic motif RRRRR(59)DT, suggesting the involvement of proprotein convertase (PC) family members in this process. METHODS AND RESULTS: Using cell transfection experiments, the processing of preprohepcidin in the human hepatocyte line Huh-7 was found to be inhibited by the Furin inhibitors serpin alpha1-antitrypsin (alpha1-PDX) and prosegment preproFurin (ppFurin). Site-directed mutagenesis analysis confirmed the RRRRR(59)DT preprohepcidin cleavage site. In parallel, the lack of preprohepcidin processing found in the PC activity-deficient cell line LoVo was restored by the expression of Furin, paired basic amino acid cleaving enzyme 4 (PACE4), PC5 or PC7. This finding is consistent with the in vitro digestions of a synthetic peptide mimicking the cleavage site of preprohepcidin. In addition, during mouse embryonic development the major expression of hepcidin found in the liver coincided with that of Furin. While hepcidin induces the degradation of the iron transporter ferroportin, its RRRRR(59) to SSSSS(59) mutant is not active. CONCLUSIONS: These results demonstrate the key role of the convertases Furin, PACE4, PC5 and/or PC7 in the generation and secretion of active hepcidin and suggest that the control of hepcidin processing as a potential therapeutic/diagnostic strategy in hepcidin-related disorders such as haemochromatosis, inflammatory diseases, anaemia and cancer.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Furina/metabolismo , Fígado/patologia , Neoplasias/patologia , Pró-Proteína Convertase 5/metabolismo , Pró-Proteína Convertases/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular/metabolismo , Furina/antagonistas & inibidores , Furina/genética , Hepcidinas , Humanos , Fígado/metabolismo , Camundongos , Mutagênese Sítio-Dirigida/métodos , Neoplasias/metabolismo , Pró-Proteína Convertase 5/genética , Pró-Proteína Convertases/genética , Serina Endopeptidases/metabolismo , Transfecção , alfa 1-Antitripsina/farmacologiaRESUMO
Infectious HIV-1 requires gp160 cleavage by furin at the REKR511 downward arrow motif (site1) into the gp120/gp41 complex, whereas the KAKR503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the present extended sequences require heparin for optimal processing. Our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the REKR sequence, which is only then optimally cleaved by furin.
Assuntos
Furina/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Heparina/farmacologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Proteína gp160 do Envelope de HIV/química , Humanos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Especificidade por Substrato/efeitos dos fármacosRESUMO
The processing of many peptides for their maturation in target tissue depends upon the presence of sorting receptor. Several previous studies have predicted that carboxypeptidase-E (CPE), prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2) may function as sorting elements for somatostatin (SST) for its maturation and processing to appropriate targets. However, nothing is currently known about whether brain, neuronal culture or even endocrine cells express SST, CPE, PC1 and PC2 and exhibit colocalization. Accordingly, in the present study using peroxidase immunohistochemistry, double-labeled indirect immunofluorescence immunohistochemistry and Western blot analysis, we mapped the distributional pattern of SST, CPE, PC1 and PC2 in different rat brain regions. Additionally, we also determined the colocalization of SST with CPE, PC1 and PC2 as well as colocalization of CPE with PC1 and PC2. The localization of SST, CPE, PC1 and PC2 reveals a distinct and region specific distribution pattern in the rat brain. Using an indirect double-label immunofluorescence method we observed selective neuron specific colocalization in a region specific manner in cortex, striatum and hippocampus. These studies provide the first evidence for colocalization between SST, CPE, PC1 and PC2 as well as CPE with PC1 and PC2. SST in cerebral cortex colocalized in pyramidal and non-pyramidal neurons with CPE, PC1 and PC2. Most importantly, in striatum and hippocampus colocalization was mostly observed selectively and preferentially in interneurons. CPE is also colocalized with PC1 and PC2 in a region specific manner. The data presented here provide a new insight into the distribution and colocalization of SST, CPE, PC1 and PC2 in rat brain. Taken together, our data anticipate the possibility that CPE, PC1 and PC2 might be potential target for the maturation of SST.
Assuntos
Química Encefálica/fisiologia , Carboxipeptidase H/metabolismo , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/metabolismo , Somatostatina/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Encéfalo/anatomia & histologia , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Hipocampo/enzimologia , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Neostriado/enzimologia , Neostriado/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.
Assuntos
Doença de Alzheimer/metabolismo , Dinorfinas/biossíntese , Endorfinas/biossíntese , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Dinorfinas/genética , Endorfinas/genética , Feminino , Humanos , Masculino , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Peptídeos Opioides/biossíntese , Peptídeos Opioides/genética , Regulação para Cima/fisiologia , NociceptinaRESUMO
Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH2-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST[1-10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys13 followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST[1-10] production. Since the prosomatostatin sequence R8-Q9-F10-L11 \ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST[1-10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST[1-10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST[1-10] required the RXRXXL motif. However, this NH2-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.
Assuntos
Pró-Proteína Convertases/farmacologia , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/farmacologia , Somatostatina/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Células COS , Cromatografia Líquida de Alta Pressão , Cricetinae , Humanos , Rim/metabolismo , Mutagênese , Mutação/genética , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Radioimunoensaio , Somatostatina/genética , Vaccinia virus/genéticaRESUMO
A somatostatin deficit occurs in the cerebral cortex of Alzheimer's disease patients without a major loss in somatostatin-containing neurons. This deficit could be related to a reduction in the rate of proteolytic processing of peptide precursors. Since the two proprotein convertases (PC)1 and PC2 are responsible for the processing of neuropeptide precursors directed to the regulated secretory pathway, we examined whether they are involved first in the proteolytic processing of prosomatostatin in mouse and human brain and secondly in somatostatin defect associated with Alzheimer's disease. By size exclusion chromatography, the cleavage of prosomatostatin to somatostatin-14 is almost totally abolished in the cortex of PC2 null mice, while the proportions of prosomatostatin and somatostatin-28 are increased. By immunohistochemistry, PC1 and PC2 were localized in many neuronal elements in human frontal and temporal cortex. The convertases levels were quantified by Western blot, as well as the protein 7B2 which is required for the production of active PC2. No significant change in PC1 levels was observed in Alzheimer's disease. In contrast, a marked decrease in the ratio of the PC2 precursor to the total enzymatic pool was observed in the frontal cortex of Alzheimer patients. This decrease coincides with an increase in the binding protein 7B2. However, the content and enzymatic activity of the PC2 mature form were similar in Alzheimer patients and controls. Therefore, the cortical somatostatin defect is not due to convertase alteration occuring during Alzheimer's disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimer's disease.
Assuntos
Doença de Alzheimer/enzimologia , Pró-Proteína Convertase 2/fisiologia , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Somatostatina/biossíntese , Somatostatina/deficiência , Somatostatina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Análise de Variância , Animais , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Feminino , Humanos , Modelos Lineares , Masculino , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 2/deficiência , Pró-Proteína Convertase 2/genética , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional/genética , Ratos , Ratos Sprague-Dawley , Somatostatina/genéticaRESUMO
The Portland alpha1-antitrypsin variant (alpha1-PDX) inhibits gp160 cleavage into gp120 and gp41 by different prohormone convertases (PCs) including furin, PC5 and PC7. Jurkat cells stably transfected with this inhibitor (J-PDX cells) and, as controls, Jurkat cells transfected with the empty vector (J-pcDNA3) were tested for their susceptibility to HIV-1 infection. We found that HIV-1 replication was significantly impaired in J-PDX cells. However, the analysis of the infectivity of HIV-1 viruses produced in J-PDX cells on different days during the infection indicated that they recovered infectivity starting from 13 days post-infection. The sequencing of viruses collected earlier and later from J-PDX cells revealed no mutations in envelope-glycoprotein precursor (Env) maturation sites or in the N-terminal sequence of gp41 fusion peptide, which plays a key role in membrane fusion. Although conserved mutations were detected at the C-terminus of the gp41 fusion peptide and ectodomain, the replication of mutant HIV-1 viruses produced on day 20 in J-PDX cells was inhibited at a similar level to wild-type viruses after a second passage in J-PDX cells. We then investigated the expression of the alpha1-PDX protein, and found that HIV-1 replication activated its proteolysis since the 54 kDa cleaved form became predominant later on in the infection. In contrast, the expression of PC7, a protein that is transported through the secretory pathway, was unaltered in HIV-1 infected cells. We conclude that recovered HIV-1 infectivity in J-PDX cells was due to a loss of alpha1-PDX activity via its extensive processing by intracellular proteases that cleave it through the substrate pathway.
Assuntos
HIV-1/fisiologia , Replicação Viral , alfa 1-Antitripsina/farmacologia , Western Blotting , Linfócitos T CD4-Positivos , Sequência Conservada , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Células Jurkat , Mutação , Peptídeos/química , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
We investigated the effects of alpha1-antitrypsine Portland variant (alpha1-PDX) and decanoylRVKRchloromethylketone (decRVKRcmk) on HIV-2(ROD) replication in the Jurkat lymphoblastoid cell line. To this end, cells were stably transfected with the alpha1-PDX (J-PDX) and used as targets for HIV-2(ROD) infection. Controls were prepared with the empty vector (J-pcDNA3). HIV-2(ROD) and HIV-1(LAI) replications were significantly inhibited and delayed in the presence of the alpha1-PDX protein. When decRVKRcmk was used at 35 microM, inhibition rates were 70-80% for HIV-2(ROD) and HIV-1(LAI), while total inhibition occurred at 70 microM. Control peptides consisting of decanoylRVKR and acetylYVADcmk had no effect. In the presence of the alpha1-PDX or the decRVKRcmk at 35 microM, the infectivity of HIV-2(ROD) and HIV-1(LAI) produced was 3-4-fold lower. Both molecules inhibited syncytium formation by HIV-2(ROD) and HIV-1(LAI) to a considerable extent. Finally, the inhibition of viral replication was correlated with the ability of the decRVKRcmk at 35 and 70 microM and of the alpha1-PDX, to inhibit the processing of envelope glycoprotein precursors. The alpha1-PDX protein and the decRVKRcmk peptide at 35 microM inhibited HIV-2 and HIV-1 to a similar level suggesting that identical or closely related endoproteases are involved in the maturation of their envelope glycoprotein precursors into surface and transmembrane glycoproteins. The significant inhibition observed with alpha1-PDX indicates that furin or furin-like endoproteases appear to play a major role in the maturation process.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Peptídeos/farmacologia , Replicação Viral/efeitos dos fármacos , alfa 1-Antitripsina/farmacologia , Clorometilcetonas de Aminoácidos , Sequência de Aminoácidos , Relação Dose-Resposta a Droga , Produtos do Gene env/metabolismo , Células Gigantes/efeitos dos fármacos , Células Gigantes/virologia , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Humanos , Células Jurkat , Peptídeos/química , Precursores de Proteínas/metabolismo , Montagem de Vírus/efeitos dos fármacos , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
The surface glycoprotein of the Lassa virus, a member of the arenaviridae family, is synthesized as a 76-kDa precursor (GP-C) that is posttranslationally cleaved into an N-terminal 44-kDa subunit and a C-terminal membrane-anchored 36-kDa subunit. Cleavage occurs at the C-terminal end of the unusual recognition motif R-R-L-L. We show here that GP-C is cleaved in the endoplasmic reticulum by the cellular subtilase SKI-1/S1P, an enzyme that has so far been observed to be involved in cholesterol metabolism. Furthermore, we present evidence that only cleaved glycoprotein is incorporated into virions and that this is necessary for the formation of infectious virus. To our knowledge, there have been no previous reports of this type of viral glycoprotein processing, one that may be an interesting target for antiviral therapy.
Assuntos
Pró-Proteína Convertases , Precursores de Proteínas/metabolismo , Serina Endopeptidases/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Células CHO , Calcimicina/farmacologia , Embrião de Galinha , Chlorocebus aethiops , Cricetinae , Vírus Lassa/efeitos dos fármacos , Vírus Lassa/metabolismo , Transfecção , Células VeroRESUMO
7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistemas Neurossecretores/fisiologia , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Humanos , Invertebrados , Mamíferos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteína Secretora Neuroendócrina 7B2 , Hormônios Hipofisários/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , VertebradosRESUMO
Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.
Assuntos
Precursores Enzimáticos/antagonistas & inibidores , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/química , Precursores de Proteínas/farmacologia , Subtilisinas/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Sequência Conservada , Humanos , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Conformação Proteica , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-AtividadeRESUMO
Proprotein convertases (PCs) of the subtilisin/kexin family are responsible for the activation of prohormones, protrophic factors, and their receptors. We sought to determine whether loss of PC-mediated activities might affect the malignant phenotypes of cancer cells. Stable transfectants of alpha(1)-antitrypsin Portland (alpha(1)-PDX) cDNA, coding for a potent PC inhibitor, were analyzed in model HT-29 cells (HT-29/PDX) and in other cell lines. Expression of alpha(1)-PDX resulted in a proinsulin-like growth factor-1 receptor (pro-IGF-1R) processing blockade, hence inhibiting the ability of exogenous IGF-1 to induce tyrosine phosphorylation of its beta-subunit and insulin-related substrate-1. Coexpression of IGF-1R with four different PCs or the novel convertase SKI-1 in the furin-defective LoVo-C5 cells demonstrated that pro-IGF-1R ( approximately 200 kDa) cleavage into IGF-1R (beta-subunit, approximately 105 kDa) can be achieved by furin and PC5A, but not by PACE4, PC7, or SKI-1. Expression of alpha(1)-PDX resulted in reduction of DNA synthesis and in anchorage-independent growth. Following serum deprivation, the alpha(1)-PDX transfectants exhibited an enhanced apoptotic phenotype and were insensitive to IGF-1-mediated [(3)H]thymidine incorporation and protection against apoptosis. These cells showed reduced invasiveness that paralleled decreased mRNA levels of urokinase-type plasminogen activator and its receptor, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. Comparative subcutaneous inoculation of cells in nude mice revealed that animals injected with HT-29/PDX cells exhibited delayed and lower incidence of tumor development as well as reduced tumor size. Immunohistochemical analysis of CD31 antigen expression, a marker of endothelial cells, revealed reduced HT-29/PDX tumor vascularization. These findings indicate that PCs actively contribute to the growth and malignant phenotypes of HT-29 tumors, suggesting that PC inhibition strategies may be a useful adduct to the arsenal of colorectal anticancer gene therapies.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Fator de Crescimento Insulin-Like I/fisiologia , Precursores de Proteínas/metabolismo , Receptor IGF Tipo 1/metabolismo , alfa 1-Antitripsina/farmacologia , Animais , Apoptose , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Células HT29 , Humanos , Imuno-Histoquímica , Proteínas Substratos do Receptor de Insulina , Masculino , Camundongos , Invasividade Neoplásica , Fosfoproteínas/metabolismo , Fosforilação , Tirosina/metabolismoRESUMO
The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N-terminal, 81-residue inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep 'autocatalytic' removal of the prodomain at the C-terminal side of the two consensus sites, R(78)-T-K-R(81) approximately and R(44)-G-V-T-K-R(49) approximately. The furin-mediated cleavage at R(44)-G-V-T-K-R(49) approximately, in particular, is significantly accelerated in an 'acidic' environment. Here, we show that under neutral pH conditions, the inhibitory prodomain of furin is partially folded and undergoes conformational exchanges as indicated by extensive broadening of the NMR spectra. Presence of many ring-current shifted methyl resonances suggests that the partially folded state of the prodomain may still possess a 'semirigid' protein core with specific packing interactions among amino acid side chains. Measurements of the hydrodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformational stability of the prodomain appears to be pH sensitive, in that the prodomain undergoes an unfolding transition towards acidic conditions. Our NMR analyses establish that the acid-induced unfolding is mainly experienced by the residues from the C-terminal half of the prodomain (residues R(44)-R(81)) that contains the two furin cleavage sites. A 38-residue peptide fragment derived from the entire pH-sensitive C-terminal region (residues R(44)-R(81)) does not exhibit any exchange-induced line broadening and adopts flexible conformations. We propose that at neutral pH, the cleavage site R(44)-G-V-T-K-R(49) approximately is buried within the protein core that is formed in part by residues from the N-terminal region, and that the cleavage site becomes exposed under acidic conditions, leading to a facile cleavage by the furin enzyme.
Assuntos
Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Dobramento de Proteína , Subtilisinas/química , Subtilisinas/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática , Precursores Enzimáticos/isolamento & purificação , Estabilidade Enzimática , Furina , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Maleabilidade , Desnaturação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Subtilisinas/isolamento & purificaçãoRESUMO
Fluorogenic peptides encompassing the processing sites of envelope glycoproteins of the infectious influenza A Hong Kong virus (HKV), Ebola virus (EBOV) and respiratory syncytial virus (RSV) were tested for cleavage by soluble recombinants of the proprotein convertases furin, PC5 and PC7. Kinetic studies with these intramolecularly quenched fluorogenic peptides revealed selective cleavages at the physiological dibasic sites. The HKV peptide is cleaved by both furin and PC5 with similar efficacy; in comparison, PC7 cleaves this substrate poorly. In contrast with the basic tetrapeptide insertion within the haemagglutinin sequence of HKV, two other dipeptide insertions revealed a poorer cleavage with a similar rank order of potency. These results demonstrate that the N-terminal RERR insertion to the wild-type avian RKKR downward arrow sequence is functionally significant, and suggest that the approx. 5-fold increase in cleavage efficacy contributes to the high infectivity of the H5N1 virus subtype. With regard to RSV peptide processing, PC7 is twice as effective as PC5 and furin. The EBOV peptide was processed with similar efficiency by the three enzymes. Our observations that all of these cleavages can be effectively inhibited by a plant andrographolide derivative at 250 microM or less might aid in the design of potent convertase inhibitors as alternative antiviral therapies.
Assuntos
Ebolavirus/metabolismo , Vírus da Influenza A/metabolismo , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Subtilisinas/metabolismo , Sequência de Aminoácidos , Corantes Fluorescentes/metabolismo , Furina , Hidrólise , Cinética , Peptídeos/química , Proteínas Recombinantes/metabolismo , Proteínas Virais/metabolismoRESUMO
Suckling increases preproTRH messenger RNA in hypothalamic paraventricular neurons (PVN) and also markedly increases TRH release during the first period of lactation. Whether lactation alters preproTRH processing resulting in the generation of novel proTRH-derived peptides that may be involved in the regulation of PRL secretion lactation is not known. Therefore, in the present study we determine whether some other peptides derived from proTRH potentially contribute to lactation-induced PRL secretion. We have recently demonstrated that two members of the family of prohormone convertases PC1 and PC2 play a significant role in proTRH processing. PC1 is the major contributor in proTRH processing, whereas PC2 may have a specific role in cleaving TRH from its extended forms. In this study, we used a recombinant vaccinia virus system to coexpress rat preproTRH complementary DNA with PC1, PC2, and the neuropeptide 7B2 in GH4C1 cells (somatomammothophs, rat). We found that two novel peptides, preproTRH(178-184) (pFQ(7)), and preproTRH(186-199) (pSE(14)), were formed after the cleavage of their precursor preproTRH(178-199) (pFE(22)) by only PC2. Their formation was confirmed by microsequence analysis. Anatomical analyses revealed that these peptides are also found in the rat PVN. In addition, we found that pFE(22), pSE(14) and pFQ(7) produced a dose-dependent release of PRL from primary cultures of pituitary cells compared with one of the well studied secretagogues of PRL, TRH. To establish whether these peptides might play a role in vivo in the regulation of PRL release, we took rat litters on postnatal day 4, separated the pups from their mothers for 6 h, and then reunited the pups and mothers for 45 min. At the end of this period, the mothers were killed, acidic extracts of microdissected PVN were prepared and subjected to SDS-PAGE, followed by slicing and analysis by pFE(22) RIA. Forty-five minutes of suckling induced a marked 6-fold increase in serum levels of PRL. In addition, PVN levels of pFE(22) and pSE(14) increased approximately 5-fold during the same period in the acutely suckling females. Lactating animals that were separated from their litters and never reunited with their pups had low levels of PRL, and pFE(22) and pSE(14). These data provide the first evidence for alterations in proTRH processing in the PVN during lactation and suggest that the products of this altered processing may play a physiological role in the regulation of PRL secretion.
Assuntos
Lactação/fisiologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Proteína Convertase 1 , Precursores de Proteínas/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Animais , Ácido Aspártico Endopeptidases/fisiologia , Células Cultivadas , Feminino , Camundongos , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Pró-Proteína Convertase 2 , Pró-Proteína Convertases , Ratos , Ratos Sprague-Dawley , Subtilisinas/fisiologiaRESUMO
We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNF-encoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr(57)- downward arrow-Ser-Leu site. Cleavage is abolished when Arg(54) is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with alpha(1)-PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Embrião de Mamíferos , Glicosídeo Hidrolases , Glicosilação , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuroglia/metabolismo , Fosforilação , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Receptor trkB/efeitos dos fármacos , Receptor trkB/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transfecção , Vaccinia virus/genéticaRESUMO
Processing of the beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretases generates the amyloidogenic peptide Abeta, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the beta-secretase beta-amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant betaAPP(sw) beta-secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylation-dependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the alpha-secretase product APPsalpha. Although there is little increase in the generation of Abeta by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced Abeta levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of betaAPP.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Processamento de Proteína Pós-Traducional , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Endopeptidases , Humanos , CamundongosRESUMO
Transforming growth factor (TGF)-beta1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-beta1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-beta1 convertase. Even though six members of the PC family have the ability to cleave TGF-beta1, ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-beta1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-beta1. Moreover, both furin and TGF-beta are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-beta1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-beta system and should facilitate the development of therapeutically useful TGF-beta inhibitors.