RESUMEN
Inhibins are members of the transforming growth factor-ß family, composed of a common α-subunit disulfide-linked to 1 of 2 ß-subunits (ßA in inhibin A or ßB in inhibin B). Gonadal-derived inhibin A and B act in an endocrine manner to suppress the synthesis of follicle-stimulating hormone (FSH) by pituitary gonadotrope cells. Roles for inhibins beyond the pituitary, however, have proven difficult to delineate because deletion of the inhibin α-subunit gene (Inha) results in unconstrained expression of activin A and activin B (homodimers of inhibin ß-subunits), which contribute to gonadal tumorigenesis and lethal cachectic wasting. Here, we generated mice with a single point mutation (Arg233Ala) in Inha that prevents proteolytic processing and the formation of bioactive inhibin. In vitro, this mutation blocked inhibin maturation and bioactivity, without perturbing activin production. Serum FSH levels were elevated 2- to 3-fold in InhaR233A/R233A mice due to the loss of negative feedback from inhibins, but no pathological increase in circulating activins was observed. While inactivation of inhibin A and B had no discernible effect on male reproduction, female InhaR233A/R233A mice had increased FSH-dependent follicle development and enhanced natural ovulation rates. Nevertheless, inhibin inactivation resulted in significant embryo-fetal resorptions and severe subfertility and was associated with disrupted maternal ovarian function. Intriguingly, heterozygous Inha+/R233A females had significantly enhanced fecundity, relative to wild-type littermates. These studies have revealed novel effects of inhibins in the establishment and maintenance of pregnancy and demonstrated that partial inactivation of inhibin A/B is an attractive approach for enhancing female fertility.
Asunto(s)
Gonadotrofos , Inhibinas , Activinas/metabolismo , Animales , Femenino , Hormona Folículo Estimulante/metabolismo , Gonadotrofos/metabolismo , Inhibinas/genética , Inhibinas/metabolismo , Masculino , Ratones , Ovario/metabolismo , Hipófisis/metabolismo , EmbarazoRESUMEN
Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing ß-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of ß-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin ßâA-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this ßâA-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin ßâB-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.
Asunto(s)
Inhibinas , Ingeniería de Proteínas/métodos , Femenino , Células HEK293 , Humanos , Inhibinas/genética , Inhibinas/aislamiento & purificación , Inhibinas/metabolismo , Inhibinas/farmacología , Proteínas de la Membrana , Modelos Moleculares , Mutagénesis/fisiología , Ovario/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Multimerización de Proteína/genética , Estructura Cuaternaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Subunidades de Proteína/farmacología , Proteínas de Saccharomyces cerevisiae , TransfecciónRESUMEN
Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are co-expressed exclusively in oocytes throughout most of folliculogenesis and play central roles in controlling ovarian physiology. Although both growth factors exist as homodimers, recent evidence indicates that GDF9 and BMP15 can also heterodimerize to form the potent growth factor cumulin. Within the cumulin complex, BMP15 "activates" latent GDF9, enabling potent signaling in granulosa cells via type I receptors (i.e. activin receptor-like kinase-4/5 (ALK4/5)) and SMAD2/3 transcription factors. In the cumulin heterodimer, two distinct type I receptor interfaces are formed compared with homodimeric GDF9 and BMP15. Previous studies have highlighted the potential of cumulin to improve treatment of female infertility, but, as a noncovalent heterodimer, cumulin is difficult to produce and purify without contaminating GDF9 and BMP15 homodimers. In this study we addressed this challenge by focusing on the cumulin interface formed by the helix of the GDF9 chain and the fingers of the BMP15 chain. We demonstrate that unique BMP15 finger residues at this site (Arg301, Gly304, His307, and Met369) enable potent activation of the SMAD2/3 pathway. Incorporating these BMP15 residues into latent GDF9 generated a highly potent growth factor, called hereafter Super-GDF9. Super-GDF9 was >1000-fold more potent than WT human GDF9 and 4-fold more potent than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Our demonstration that Super-GDF9 can effectively promote mouse cumulus cell expansion and improve oocyte quality in vitro represents a potential solution to the current challenges of producing and purifying intact cumulin.
Asunto(s)
Factor 9 de Diferenciación de Crecimiento/metabolismo , Oocitos/metabolismo , Animales , Proteína Morfogenética Ósea 15/genética , Proteína Morfogenética Ósea 15/metabolismo , Línea Celular Tumoral , Femenino , Variación Genética/genética , Factor 9 de Diferenciación de Crecimiento/genética , Humanos , Ratones , Modelos Moleculares , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismoRESUMEN
Gonadal-derived inhibin A and B are essential factors in mammalian reproduction, negatively regulating pituitary production of FSH. Inhibins are synthesized as heterodimers of α- and ß-subunits, each comprising an N-terminal pro- and C-terminal mature domain. After dimerization, the inhibin α- and ß-subunit prodomains are enzymatically cleaved from the mature domains at consensus RXXR sites (site1). Interestingly, the inhibin α-subunit is a unique TGF-ß ligand, comprising a second cleavage site (site2) within its prodomain. Cleavage at site2 in the inhibin α-subunit prodomain releases a 43-amino acid proα-peptide. We aimed to determine the influence of the proα-peptide on inhibin synthesis and bioactivity. Blocking proα-peptide release by silencing cleavage site2 (Arg56-Arg61) inhibited both inhibin A and B synthesis. Ligand blot analysis and solid-phase binding assays indicated that the proα-peptide binds specifically to a mature 30-kDa inhibin (mean Kd 86 nM) but was unable to bind related activins. The proα-peptide suppressed inhibin A and B bioactivity in primary rat pituitary cell cultures. Mechanistically, the proα-peptide blocked inhibin A binding to its coreceptor, betaglycan (IC50 131 nM), and the subsequent sequestration of the activin type II receptor (IC50 156 nM), which underscores inhibin's biological activity. Based on the sequential mutations across the inhibin α-subunit, the proα-peptide binding site was localized to residues Arg341-Thr354, corresponding directly to the betaglycan binding region. Together our findings indicate that the proα-peptide limits the synthesis and bioactivity of inhibins.
Asunto(s)
Inhibinas/biosíntesis , Inhibinas/farmacología , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/química , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Humanos , Inhibinas/antagonistas & inhibidores , Inhibinas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteoglicanos/metabolismo , Ratas , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Inhibin ELISAs are used in monitoring aspects of reproductive function, however these assays are based on the measurements of the mature 30kDa inhibin forms and not precursor forms. In conventional ELISA formats, the 105kDa unprocessed 'Pro-inhibin' forms are immunologically inactive, but the immunoactivity can be recovered in the presence of detergents. The immunoactivity of Pro-inhibin forms was assessed in the presence of a range of detergents utilising antibodies to the α-, ßA- and ßB-subunits of inhibin. In contrast to mature forms, unprocessed inhibin forms showed a 10-40 fold increase in inhibin A and total inhibin immunoactivities under optimised detergent (0.5% SDS/2% Triton X-100) conditions. The suppressed immunoactivity of the Pro-inhibin forms in these immunoassays was attributed to steric hindrance by the respective ßA- and α-subunit prodomains. This study details a detergent-based immunoassay that allows detection of previously undetectable precursor inhibin forms.
Asunto(s)
Detergentes/química , Inhibinas/química , Tampones (Química) , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Líquido Folicular/metabolismo , Células HEK293 , Humanos , Inhibinas/aislamiento & purificación , Inhibinas/metabolismo , Octoxinol/química , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/aislamiento & purificación , Precursores de Proteínas/metabolismo , Dodecil Sulfato de Sodio/químicaRESUMEN
Growth differentiation factor 9 (GDF9) controls granulosa cell growth and differentiation during early ovarian folliculogenesis and regulates cumulus cell function and ovulation rate in the later stages of this process. Similar to other TGF-ß superfamily ligands, GDF9 is secreted from the oocyte in a noncovalent complex with its prodomain. In this study, we show that prodomain interactions differentially regulate the activity of GDF9 across species, such that murine (m) GDF9 is secreted in an active form, whereas human (h) GDF9 is latent. To understand this distinction, we used site-directed mutagenesis to introduce nonconserved mGDF9 residues into the pro- and mature domains of hGDF9. Activity-based screens of the resultant mutants indicated that a single mature domain residue (Gly(391)) confers latency to hGDF9. Gly(391) forms part of the type I receptor binding site on hGDF9, and this residue is present in all species except mouse, rat, hamster, galago, and possum, in which it is substituted with an arginine. In an adrenocortical cell luciferase assay, hGDF9 (Gly(391)Arg) had similar activity to mGDF9 (EC(50) 55 ng/ml vs. 28 ng/ml, respectively), whereas wild-type hGDF9 was inactive. hGDF9 (Gly(391)Arg) was also a potent stimulator of murine granulosa cell proliferation (EC(50) 52 ng/ml). An arginine at position 391 increases the affinity of GDF9 for its signaling receptors, enabling it to be secreted in an active form. This important species difference in the activation status of GDF9 may contribute to the variation observed in follicular development, ovulation rate, and fecundity between mammals.
Asunto(s)
Factor 9 de Diferenciación de Crecimiento/metabolismo , Mutación Puntual , Corteza Suprarrenal/citología , Secuencia de Aminoácidos , Animales , Arginina/química , Proliferación Celular , Femenino , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ovulación , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Elevated activin A levels in inhibin-deficient mice promote the development of gonadal tumors and induce cachexia by reducing muscle, liver, stomach, and fat mass. Because activin A is an important regulator of tissue growth, inhibiting the actions of this TGFß family ligand may halt or reverse pathology in diseased tissues. In this study, we modified the activin A propeptide to generate a specific activin antagonist. Propeptides mediate the synthesis and secretion of all TGFß ligands and, for some family members (e.g. TGFß1), bind the mature growth factor with high enough affinity to confer latency. By linking the C-terminal region of the TGFß1 propeptide to the N-terminal region of the activin A propeptide, we generated a chimeric molecule [activin/TGFß1 propeptide (AT propeptide)] with increased affinity for activin A. The AT propeptide was 30-fold more potent than the activin A propeptide at suppressing activin-induced FSH release by LßT2 pituitary gonadotrope cells. Binding of the AT propeptide to activin A shields the type II receptor binding site, thereby reducing Smad2 phosphorylation and downstream signaling. In comparison with the commonly used activin antagonists, follistatin (IC(50) 0.42 nM), soluble activin type II receptor A-Fc (IC(50) 0.47 nM), and soluble activin type II receptor B-Fc (IC(50) 0.91 nM), the AT propeptide (IC(50) 2.6 nM) was slightly less potent. However, it was more specific, inhibiting activin A and activin B (IC(50) 10.26 nM) but not the closely related ligands, myostatin and growth differentiation factor-11. As such, the AT propeptide represents the first specific activin antagonist, and it should be an effective reagent for blocking activin actions in vivo.
Asunto(s)
Activinas/antagonistas & inhibidores , Precursores de Proteínas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Factor de Crecimiento Transformador beta1/biosíntesis , Activinas/biosíntesis , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Precursores de Proteínas/farmacología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Transforming growth factor-beta1 (TGF-beta1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-beta1 propeptide (latency-associated peptide (LAP)), and latent TGF-beta-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-beta1 complex. Initially, hydrophobic LAP residues (Ile(53), Leu(54), Leu(57), and Leu(59)), which form a contiguous epitope on one surface of an amphipathic alpha-helix, interact with mature TGF-beta1 to form the small latent complex. TGF-beta1 binding is predicted to alter LAP conformation, exposing ionic residues (Arg(45), Arg(50), Lys(56), and Arg(58)) on the other side of the alpha-helix, which form the binding site for latent TGF-beta-binding proteins. The stability of the resultant large latent complex is dependent upon covalent dimerization of LAP, which is facilitated by key residues (Phe(198), Asp(199), Val(200), Leu(208), Phe(217), and Leu(219)) at the dimer interface. Significantly, genetic mutations in LAP (e.g. R218H) that cause the rare bone disorder Camurati-Engelmann disease disrupted dimerization and reduced the stability of the latent TGF-beta1 complex.
Asunto(s)
Proteínas de Unión a TGF-beta Latente/química , Péptidos/química , Factor de Crecimiento Transformador beta1/metabolismo , Secuencia de Aminoácidos , Bioensayo , Síndrome de Camurati-Engelmann/metabolismo , Medios de Cultivo Condicionados/farmacología , Dimerización , Matriz Extracelular/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Estructura Terciaria de Proteína , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The assembly and secretion of transforming growth factor beta superfamily ligands is dependent upon non-covalent interactions between their pro- and mature domains. Despite the importance of this interaction, little is known regarding the underlying regulatory mechanisms. In this study, the binding interface between the pro- and mature domains of the inhibin alpha-subunit was characterized using in vitro mutagenesis. Three hydrophobic residues near the N terminus of the prodomain (Leu(30), Phe(37), Leu(41)) were identified that, when mutated to alanine, disrupted heterodimer assembly and secretion. It is postulated that these residues mediate dimerization by interacting non-covalently with hydrophobic residues (Phe(271), Ile(280), Pro(283), Leu(338), and Val(340)) on the outer convex surface of the mature alpha-subunit. Homology modeling indicated that these mature residues are located at the interface between two beta-sheets of the alpha-subunit and that their side chains form a hydrophobic packing core. Mutation of these residues likely disturbs the conformation of this region, thereby disrupting non-covalent interactions with the prodomain. A similar hydrophobic interface was identified spanning the pro- and mature domains of the inhibin beta(A)-subunit. Mutation of key residues, including Ile(62), Leu(66), Phe(329), and Pro(341), across this interface was disruptive for the production of both inhibin A and activin A. In addition, mutation of Ile(62) and Leu(66) in the beta(A)-propeptide reduced its ability to bind, or inhibit the activity of, activin A. Conservation of the identified hydrophobic motifs in the pro- and mature domains of other transforming growth factor beta superfamily ligands suggests that we have identified a common biosynthetic pathway governing dimer assembly.
Asunto(s)
Inhibinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inhibinas/química , Inhibinas/genética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Factor de Crecimiento Transformador beta/clasificaciónRESUMEN
Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin alpha-subunit. Homology modeling indicates that key alpha-subunit residues (Tyr(50), Val(108), Thr(111), Ser(112), Phe(118), Lys(119), and Tyr(120)) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr(111), Ser(112), and Tyr(120) to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val(108)-Tyr(120) of the inhibin alpha-subunit mediate inhibin A biological activity.
Asunto(s)
Inhibinas/antagonistas & inhibidores , Proteoglicanos/química , Receptores de Factores de Crecimiento Transformadores beta/química , Activinas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Follistatin is a potent extracellular antagonist of members of the TGFbeta superfamily that use activin type II receptors (ActRII/IIB) as part of their signaling complex. A recent crystallographic study indicates that follistatin contacts activin-A residues at both the type I (ALK4) and type II receptor binding interfaces. However, the relative contribution of these two sites on human activin-A to follistatin binding has not been determined. Residues at these sites were mutated to alanine and mutants were screened for their ability to bind follistatin and ActRII and induce FSH secretion from a gonadotrope cell line. Despite extensive mutagenesis across the type I receptor interface, activin-A affinity for follistatin was not significantly diminished. In contrast, mutagenesis of residues at the type II binding interface had pronounced effects on activin's interaction with follistatin. In particular, residues Leu92, Tyr94, Ile100, and Lys102 were critical for high-affinity follistatin binding. Interestingly, mutation of another primary determinant of ActRII/IIB binding, Ser90, did not affect follistatin affinity, suggesting that the interaction surfaces for type II receptors and follistatin were overlapping but not identical. In support, mutation of Asp95, on the opposite edge of the common ActRII/follistatin interface, was disruptive for follistatin binding without affecting ActRII/IIB interactions. Activin-S90A was able to compete with wild-type activin for follistatin binding, whereas activin-D95A, due to its 8-fold lower affinity for follistatin, is a potent activin agonist. These reagents could be used to modulate follistatin antagonism of activin and related ligands in processes such as cancer, wound healing, and reproduction.