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

项目概述：LMP-1是我们实验室发现的细胞内LIM结构域蛋白，在体外已证明能够显著提高细胞对BMP-2的反应性。本研究的长期目标是证实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的反应性的假设。具体目的是证明LMP-1通过：1)中断smurf1介导的R-Smads蛋白酶体降解（Smad1）增加对BMP-2的反应性；2)阻断Smurf1与I-Smads的相互作用（Smad6），阻断l-Smad/Smurf1介导的BMP受体蛋白酶体降解（BMPR1A）；3)阻断jab1介导的普通Smad蛋白酶体降解（Smad4）。我们的最终目标是确定LMP-1中特定的相互作用基序，从而设计一个小分子来模拟LMP-1的能力，以增强细胞对BMP-2的反应性。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、计算药物设计技术、BMP-2反应性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。高达45%的患者使用髂骨移植物可能无法实现坚实的骨融合，高达25%的患者可能在供体部位出现慢性疼痛。从这些实验中获得的信息将进一步了解细胞对骨形态发生蛋白（骨形成的关键调节因子）的反应性调节，并可能导致设计出可以大大增强bmp效能的小分子，从而使患者在临床上更负担得起bmp。

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.