Microfibrils are extracellular matrix structures that provide crucial properties to a wide variety of both elastic and non-elastic tissues. The major and best characterized component of the microfibrils is fibrillin-1, a 350 kDa glycoprotein. Mutations in fibrillin-1 are the cause of the Marfan syndrome, a dominantly inherited disorder characterized by both pleiotropic manifestations affecting the skeletal, cardiovascular and ocular systems and a wide range of severity of symptoms, both between and within families. Although numerous FBN1 mutations have been identified in patients with both Marfan syndrome and related disorders, very little is understood concerning the factors determining the resulting clinical phenotype. The associated pathology emphasizes the need to understand both the cellular processing of fibrillin-1 and the role of this processing in microfibril assembly. The long-term goal of the proposed research is to delineate the cellular processing of fibrillin-1 and to characterize the proteins involved in the processing. Based on previously published studies and preliminary data, a hypothetical model for the cellular proteolytic processing of fibrillin-1 is proposed. This model hypothesizes that fibrillin-1 is synthesized as a proprotein and directed into the endoplasmic reticulum. A chaperone protein attaches to the synthesized proprotein to prevent the premature processing of Profibrillin as it is secreted from the cell. As profibrillin is secreted or shortly thereafter, 140 residues of the carboxyl-terminal fragment are removed by a protease that is a member of the PACE (furin) family. The protease responsible for profibrillin-1 processing is located in the secretory pathway beyond the trans-Golgi network. Proteolytic removal of the carboxyl-terminal fragment is required before fibrillin-1 can assemble into microfibrils and this processing plays a role in controlling this assembly.
The specific aims test various aspects of this hypothetical model of profibrillin-1 cellular processing; specifically, delineate the carboxyl-terminal cleavage site, determine if profibrillin is associated with a chaperone protein, characterize the expression pattern and subcellular localization of PACE-like enzymes in fibroblasts and smooth muscle cells, and further identify cell strains with defects in the proteolytic processing of profibrillin-1. These results have implications for understanding both the cellular processing of profibrillin-1 and the factors that regulate the activity of the PACE family of intracellular proteases. Altogether, the proposed studies will enhance our understanding of the cell biology of fibrillin and shed new light on how defects in this system lead to disease states.
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