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

描述（由申请人提供）：项目概述：LMP-1是在我们实验室中鉴定的细胞内LIM结构域蛋白，其已证明能够显著增加体外细胞对BMP-2的反应性。该提案的长期目标是证实LMP-1通过调节对BMP-2信号传导重要的关键分子的蛋白酶体降解来调节细胞对BMP的反应性的假设。具体目的是证明LMP-1通过以下方式增加对BMP-2的响应性：1）中断Smurf 1介导的R-Smads（Smad 1）的蛋白酶体降解; 2）阻断Smurf 1与I-Smads（Smad 6）的相互作用，中断I-Smad/Smurf 1介导的BMP受体（BMPR 1A）的蛋白酶体降解; 3）中断Jab 1介导的常见Smad（Smad 4）的蛋白酶体降解。我们的最终目标是确定LMP-1中的特定相互作用基序，从而能够设计小分子来模拟LMP-1增强细胞对BMP-2的反应性的能力。所采用的方法将包括蛋白质分离、体外转录/翻译、Western印迹、艾尔莎、泛素化测定、实时RT-PCR、计算药物设计技术、BMP-2反应性的体外测试和异位骨形成的体内测试。相关性：骨科最大的临床挑战之一是无法持续生成用于脊柱融合和骨缺损重建的骨。使用髂嵴植骨可能无法在高达45%的患者中实现牢固的骨融合，高达25%的患者可能会在供区出现慢性疼痛。从这些实验中了解到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，并可能导致小分子的设计，这些小分子可以大大增强BMP的效力，从而使它们在临床上更适合患者。

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.