RESUMEN
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.
Asunto(s)
Endopeptidasas/metabolismo , Subtilisinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , ADN Recombinante , Endopeptidasas/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Furina , Regulación de la Expresión Génica , Ratones , Mutación , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Subtilisinas/genética , Transfección , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1 , Células Tumorales Cultivadas , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
Sequence analysis of the adhesion molecule E-cadherin had revealed a multibasic motif [4PArg-Gln-Lys-Arg1P], reminiscent of the minimal cleavage signal for furin, the prototype of the proprotein convertase family, and/or other members sharing similar sequence specificity. Mutation of this site was sufficient to abolish processing of E-cadherin in fibroblasts reinforcing the possibility that proprotein convertases are involved in the maturation of this adhesion molecule. Here we demonstrate that even though furin can efficiently and specifically cleave proE-cadherin in a baculovirus-based co-expression system, the furin-deficient LoVo cells were found to process endogenous E-cadherin as efficiently as normal cell lines. This suggests, for the first time, that E-cadherin is not only a substrate for furin but for other mammalian convertases sharing similar sequence specificity.
Asunto(s)
Cadherinas/biosíntesis , Procesamiento Proteico-Postraduccional , Subtilisinas/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae , Cadherinas/química , Cadherinas/metabolismo , Células Cultivadas , Furina , Humanos , Insectos , Células L , Ratones , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFbeta1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFbeta1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFbeta1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFbeta1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFbeta1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFbeta1 by coexpressing prohTGFbeta1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFbeta1 increased in a dose-dependent fashion reaching up to 7. 8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFbeta1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.
Asunto(s)
Proteínas de la Membrana , Proteínas Recombinantes/biosíntesis , Subtilisinas/biosíntesis , Transfección/métodos , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Baculoviridae/genética , Línea Celular , Furina , Vectores Genéticos , Humanos , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/genética , Spodoptera , Subtilisinas/genética , Subtilisinas/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
TGFbeta1 is known for its potent and diverse biological effects, including immune regulation, and cell growth and differentiation. We have recently shown that TGFbeta1 precursor is processed by human furin COOH-terminal to the R-H-R-R278 cleavage site to generate authentic mature TGFbeta1. In the present study, we demonstrate that steady-state furin mRNA levels are increased in rat synovial cells by 2 and 20 ng/ml TGFbeta1. Stimulation with TGFbeta1 results in a significant increase in furin mRNA levels, starting at 3 h with the peak effect observed at 12 h (2.5-fold increase +/-0.4). TGFbeta1 did not increase furin mRNA stability, and treatment of synovial cells with actinomycin D, before TGFbeta1 addition prevented the increase in fur gene expression, suggesting that the observed regulation occurs at the level of gene transcription. Treatment of synovial and NRK-49F fibroblastic cells with exogenous TGFbeta1 (5 ng/ml) or TGFbeta2 (10 ng/ml) translates into an increase in pro-TGFbeta1 processing as evidenced by the appearance of a 40-kD immunoreactive band corresponding to the TGFbeta1 NH2-terminal pro-region. Furin processing activity stimulated by TGFbeta2 correlates with significant increase in extracellular mature and heat-activable TGFbeta1 as determined by an isoform-specific ELISA assay. Taken together, these results demonstrate for the first time that TGFbeta1 upregulates gene expression of its own converting enzyme, and that this expression is translated into augmented processing of the TGFbeta1 precursor form. Such adaptive responsiveness of the TGFbeta1 convertase may represent an important aspect of TGFbeta1 bioavailibility in TGFbeta1-related processes and pathological conditions.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Subtilisinas/genética , Subtilisinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , ADN/genética , Furina , Humanos , Hibridación in Situ , Interleucina-1/farmacología , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Membrana Sinovial/citología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Proteolytic processing of the transforming growth factor beta precursor (pro-TGF beta) is an essential step in the formation of the biologically active TGF beta homodimeric protein (TGF beta). The 361-amino-acid precursor pro-TGF beta 1 has within its primary structure the R-H-R-R processing signal found in many constitutively secreted precursor proteins and potentially recognized by members of the mammalian convertase family of endoproteases. To determine whether cleavage of pro-TGF beta 1 can be achieved by the furin convertase in vitro, purified precursor was incubated in the presence of a truncated/secreted form of the enzyme. Immunoblots showed that the 55-kDa pro-TGF beta 1 was converted into the 44 and 12.5 kDa bands corresponding to the pro-region and the mature monomer, respectively. Treatment of pro-TGF beta 1 with furin resulted in a 5-fold increase in the production of biologically active TGF beta 1. Furthermore, when expressed in the furin-deficient LoVo cells, no processing of pro-TGF beta 1 was observed. In contrast, efficient processing was observed when pro-TGF beta was coexpressed with the furin convertase. Collectively, these results provide evidence that in our experimental systems the TGF beta 1 precursor is efficiently and correctly processed by human furin thus permitting release of the biologically active peptide.