LMP-1 is an intracellular LIM domain protein identified in our laboratory which has demonstrated an ability to dramatically increase cellular responsiveness to BMP-2 in vitro. The long term goals of this proposal are to confirm the hypothesis that LMP-1 modulates cellular responsiveness to BMPs through regulation of proteasomal degradation of key molecules that are important for BMP-2 signaling.
The specific aims are to demonstrate that LMP-1 increases responsiveness to BMP-2 by: 1) interrupting Smurf1-mediated proteasomal degradation of R-Smads (Smad1); 2) blocking interaction of Smurf1 with I-Smads (Smad6), interrupting l-Smad/Smurf1-mediated proteasomal degradation of the BMP receptor (BMPR1A); and, 3) interrupting Jab1-mediated proteasomal degradation of the common Smad (Smad4).
Our final aim will identify the specific interacting motifs in LMP-1, enabling design of a small molecule to mimic the ability of LMP-1 to enhance cellular responsiveness to BMP-2. The methods employed will include protein isolation, in vitro transcription/translation, Western blotting, ELSA, ubiquitination assays, real-time RT-PCR, computational drug design techniques, in vitro testing of BMP-2 responsiveness, and in vivo testing of ectopic bone formation.
One of the greatest clinical challenges in Orthopaedics is the inability to consistently generate bone for spinal fusion and bone defect reconstruction. Use of iliac crest bone graft may fail to achieve solid bony fusion in up to 45% of patients, and up to 25% may experience chronic pain at the donor site. The information learned from these experiments will further the understanding of the regulation of cellular responsiveness to bone morphogenetic proteins, the key regulators of bone formation, and potentially lead to the design of small molecules that could greatly enhance the potency of BMPs, thereby making them more clinically affordable for patients. ? ?
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