LMP-1 is a critical regulator of BMP signaling and BMP responsiveness

描述(申请人提供)：项目总结：LMP-1是我们实验室鉴定的一种细胞内LIM结构域蛋白，已证明在体外能够显著提高细胞对BMP-2的反应性。该提案的长期目标是确认LMP-1通过调节对BMP-2信号重要的关键分子的蛋白酶体降解来调节细胞对BMP的反应的假说。其具体目的是证明LMP-1通过以下途径增加对BMP-2的反应性：1)阻断SMurf1介导的R-Smad的蛋白酶体降解(Smad1)；2)阻断Smr1与I-Smads的相互作用(Smad6)，阻断L-Smad/SmRa介导的BMP受体的蛋白酶体降解(BMPR1a)；以及3)阻断Jab1介导的普通Smad的蛋白酶体降解(Smad4)。我们的最终目标将确定LMP-1中特定的相互作用基序，使设计一个小分子来模拟LMP-1的能力，以增强细胞对BMP-2的反应。所采用的方法将包括蛋白质分离、体外转录/翻译、Western blotting、ELSA、泛素化分析、实时RT-PCR、计算机药物设计技术、BMP-2体外反应测试以及体内异位骨形成测试。相关性：骨科最大的临床挑战之一是不能始终如一地为脊柱融合和骨缺损重建生成骨。使用髂骨植骨可能无法在高达45%的患者中实现坚固的骨融合，高达25%的患者可能会在供体部位经历慢性疼痛。从这些实验中获得的信息将进一步了解细胞对骨形成蛋白(骨形成的关键调节因子)的反应性调节，并可能导致设计能够极大地增强BMPs效力的小分子，从而使患者更容易负担得起它们。

DESCRIPTION (provided by applicant): PROJECT SUMMARY: 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. RELEVANCE: 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.