RESUMEN
The structure and function(s) of the very large proregions of the transforming growth factor-ß structure family are known in only a few cases. The proregion of growth and differentiation factor (GDF)5 comprises 354 residues. GDF5 therefore belongs to the group of those growth factors with the largest proregions. Here, we report a biophysical analysis of the proform (proGDF5) and the separate proregion. In the absence of the mature part, the proregion folds reversibly to form a monomeric polypeptide that is stabilized by an intramolecular disulfide bond. In the context of the mature part, i.e. in proGDF5, the proregion shows increased thermodynamic stability and contains a higher proportion of secondary structural elements than in its isolated form. A subdomain within the proregion represents a well-folded structure as monitored via biophysical analysis and NMR spectroscopy. Furthermore, two point mutations that are associated with skeletal malformations lead to reduced thermodynamic stability, which is interpreted on the basis of a homology model with the structure of the related latency-associated peptide, representing the proregion of transforming growth factor-ß1.
Asunto(s)
Factor 5 de Diferenciación de Crecimiento/química , Sustitución de Aminoácidos , Braquidactilia/genética , Dicroismo Circular , Cistina/análisis , Factor 5 de Diferenciación de Crecimiento/efectos de los fármacos , Factor 5 de Diferenciación de Crecimiento/fisiología , Calor , Humanos , Modelos Moleculares , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Mutación Puntual , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Precursores de Proteínas/química , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5(T201P) and proGDF5(L263P) revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFß1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.
Asunto(s)
Desarrollo Óseo/genética , Braquidactilia/genética , Condrogénesis/genética , Factor 5 de Diferenciación de Crecimiento/genética , Secuencia de Aminoácidos , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Huesos/embriología , Cartílago/embriología , Cartílago/crecimiento & desarrollo , Diferenciación Celular/genética , Proliferación Celular/genética , Heterocigoto , Humanos , Cariotipo , Mutación Missense , Alineación de Secuencia , Proteínas Smad/metabolismoRESUMEN
Proregions of bone morphogenetic proteins (BMPs) fulfill important biological functions as intramolecular chaperones and for extracellular targeting of the mature signal ligand. Knowledge of the structures of the proregions would contribute to a more comprehensive picture of the biological activities of the pro-forms of BMP growth factors. In this study, a protease resistant core domain of the proregions of three BMPs was identified. For a more detailed analysis, the core domain of the BMP-2 proregion was recombinantly produced. Unfolding/refolding experiments and spectroscopic analyses proved that the core domain can be obtained as a folded entity. Binding of the core domain to the mature growth factor was demonstrated by SPR. Via peptide microarray analysis, residues within the core fragment could be identified that engage in binding to the dimeric, mature domain. Our study reveals that diverse members of the BMP family share a common, independently folding core domain within the large proregions peculiar to TGF-ß superfamily members that may serve as a scaffold for folding and assembly of the dimeric proprotein complexes.