The central hypothesis of this P01 proposal is that increased TGFbeta levels play an important role in the development of bronchopulmonary dysplasia of ventilated preterm infants by interfering with the cellular growth and differentiation eents that underlie normal lung morphogenesis. The long term goal of this part of the P01 is to design a viable therapeutic agent that will modulate the signaling of TGFbeta. Based on the interpretation of a preliminary computer model of the TGFbeta type II receptors, we already have two candiate peptides that can antagonize TGFbeta signaling. Our hypothesis is that, for signaling to occur, a direct interaction is required between the TGFbeta type I and type II receptors, and that this interaction is mediated by two cysteine-cysteine bridged loops of the type II receptor. We believe that theinhibitory peptides, whose seqences correspond to these loops, modulate TGFbeta signaling by interfering with this intraction. To examine this hypothesis, we will perform a structure-function study of the extracellular part of the TGFbeta signaling complex, and develop and understanding of themolecular nature of the complex. The proposal has three specific aims as follows. (1) To construct a model three-dimensional structure of the extracellular domain of the TGFbeta ligan-receptor signaling complex. This will involve construction of models of the type I and type II receptors, wusing secondary structure prediction, homology modeling, model building, and molecular dynamics. Various models will be examined for docking of the receptors with the X-ray structures of the TGFbeta isoforms. (2) To raise polyclonal and monoclonal antibodies against peptides derived from either the ligand or the receptor, in order to validate aspects of the signaling complex model. Using our model structure, we will select appropriate peptides against which we will raise first polyclonaland then monoclonal antibodies. These antibodies will be used to probe the solvent exposre of specific sequences in the receptors and the potential role of these sequences in ligand-receptor and receptor - receptor interactions. (3) To synthesize peptide fragments that compete with TGFbeta for receptor binding sites, or interfere with receptor-receptor interactions. We already have two peptide fragments ofthe TGFbeta type II receptor that are able to inhibit TGFbeta signaling and could be lead compounds in the design of a therapeutic agent. Here we will use modified peptides, based on these lead peptides, tofurther probe the moleccular nature of the signaling complex.
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