IGFBP 4 PROTEASE AND HUMAN OSTEOBLASTS

IGFBP 4 蛋白酶和人类成骨细胞

• 批准号: 6532973
• 负责人: Xuezhong Qin
• 金额: $ 16.38万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-04 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6532973
• 关键词: affinity chromatography; bone morphogenetic proteins; conformation; crosslink; endopeptidases; enzyme activity; gel filtration chromatography; human tissue; insulinlike growth factor; ion exchange chromatography; mass spectrometry; osteoblasts; physiologic bone resorption; polymerase chain reaction; protein binding; protein degradation; protein sequence; protein structure function; proteolysis; site directed mutagenesis; tissue /cell culture; western blottings

Insulin-like growth factors (IGFs) are important stimulators of bone formation. Past findings that IGFBP-4 is a potent inhibitor of IGF action in bone cells and that IGFBP-4 production is regulated by osteoregulatory agents support our premise that IGFBP-4 is a key component of the IGF system in bone. In addition, there is evidence that IGFBP-4 concentration is regulated at the local level by mechanisms involving both synthesis and degradation. In this study, we chose to evaluate the role of IGF-II dependent IGFBP-4 protease (IGFBP-4 protease) in regulating the availability, and thus the activity of IGFBP-4 in human osteoblasts (hOBs) for the following reasons: 1) IGFBP-4 protease produced by hOBs cleaves IGFBP-4 into forms with low IGF binding affinity and thus may control IGF-II release from the IGFBP-4/IGF-II complex, 2) The activity of IGFBP-4 protease is regulated by osteoregulatory agents such as IGF-II and TGF- and is thus relevant to bone formation. 3) IGFBP-4 protease is the newest component of the IGF system and its role in hOB metabolism has not been established. The aims of this study are to evaluate the role of IGFBP-4 protease in modulating IGF action in hOBs and elucidate the mechanism involved in IGF-II enhancement of IGFBP-4 degradation. Two hypotheses will be tested: 1) IGFBP-4 protease is an important regulatory component of the IGF system in hOBs. 2) Binding of IGF-II to IGFBP-4 alters the conformation such that the protease recognition sequence in IGFBP-4 is more accessible to IGFBP-4 protease. To test hypothesis 1, we will prepare partially purified IGFBP-4 protease to identify the primary cleavage site and the protease recognition sequence. This information together with our knowledge of the localization of the IGF binding domain will be used to design IGFBP-4 analogs which are fully protease resistant (PR) but bind to IGF with similar affinity as that of the wild type (WT). Such analogs will be used to evaluate the role of IGFBP-4 protease in releasing IGF-II from IGF-II/IGFBP-4 complex thereby increasing the local mitogenic activity of the IGFs. To test hypothesis 2, we will prepare analogs that have reduced IGF-II binding affinity but retain the proteolytic domain. If IGF-II fails to enhance degradation of such analogs, such a finding would suggest that the binding of IGF-II to IGFBP-4 is essential to IGFBP-4 proteolysis. Then whether binding of IGF-II to IGFBP-4 increases the association between IGFBP-4 and IGFBP-4 protease will be determined. We anticipate that completion of these studies will lead to our greater understanding of the role of IGFBP-4 protease in the regulation of hOB proliferation and provides a basis for the design of peptide-based IGFBP-4 protease activators for osteoporosis treatment. Moreover, PR IGFBP-4 analog (if more potent than WT IGFBP-4) may have therapeutic potential in the treatment of IGF dependent cancers.

胰岛素样生长因子（IGFs）是骨形成的重要刺激因子。 过去的研究结果表明，IGFBP-4是骨细胞中IGF作用的有效抑制剂，IGFBP-4的产生受骨调节剂的调节，这支持了我们的假设，即IGFBP-4是骨中IGF系统的关键组分。 此外，有证据表明IGFBP-4浓度在局部水平上通过涉及合成和降解的机制进行调节。 在这项研究中，我们选择评估IGF-II依赖性IGFBP-4蛋白酶的作用，IGFBP-4蛋白酶）在调节人成骨细胞（hOB）中IGFBP-4的可用性，从而调节IGFBP-4的活性中的作用，原因如下：1）由hOB产生的IGFBP-4蛋白酶将IGFBP-4切割成具有低IGF结合亲和力的形式，因此可以控制IGF-II从IGFBP-4/IGF-II复合物的释放，2）IGFBP-4蛋白酶的活性受骨调节剂如IGF-II和TGF-β的调节，因此与骨形成相关。 3)IGFBP-4蛋白酶是IGF系统的最新组分，其在hOB代谢中的作用尚未确定。 本研究的目的是评估IGFBP-4蛋白酶在调节hOB中IGF作用中的作用，并阐明IGF-II增强IGFBP-4降解的机制。 将检验两个假设：1）IGFBP-4蛋白酶是hOB中IGF系统的重要调节组分。2)IGF-II与IGFBP-4的结合改变了构象，使得IGFBP-4中的蛋白酶识别序列更容易接近IGFBP-4蛋白酶。 为了检验假设1，我们将制备部分纯化的IGFBP-4蛋白酶以鉴定主要切割位点和蛋白酶识别序列。 该信息连同我们对IGF结合结构域的定位的知识将用于设计IGFBP-4类似物，其是完全蛋白酶抗性的（PR），但以与野生型（WT）相似的亲和力结合IGF。 这些类似物将用于评价IGFBP-4蛋白酶在从IGF-II/IGFBP-4复合物释放IGF-II从而增加IGF的局部促有丝分裂活性中的作用。 为了检验假设2，我们将制备具有降低的IGF-II结合亲和力但保留蛋白水解结构域的类似物。 如果IGF-II不能增强这些类似物的降解，则这一发现表明IGF-II与IGFBP-4的结合对于IGFBP-4蛋白水解是必需的。 然后确定IGF-II与IGFBP-4的结合是否增加IGFBP-4和IGFBP-4蛋白酶之间的结合。 我们预期这些研究的完成将使我们更好地理解IGFBP-4蛋白酶在hOB增殖调节中的作用，并为设计基于肽的IGFBP-4蛋白酶激活剂治疗骨质疏松症提供基础。 此外，PR IGFBP-4类似物（如果比WT IGFBP-4更有效）可能在治疗IGF依赖性癌症中具有治疗潜力。

项目成果

Covalent interaction between proform of eosinophil major basic protein (proMBP) and pregnancy-associated plasma protein-A (PAPP-A) is a cell-mediated event and required for proMBP inhibition of the catalytic activity of PAPP-A.

嗜酸性粒细胞主要碱性蛋白原体 (proMBP) 与妊娠相关血浆蛋白 A (PAPP-A) 之间的共价相互作用是细胞介导的事件，是 proMBP 抑制 PAPP-A 催化活性所必需的。

• DOI: 10.1016/j.abb.2004.01.005
• 发表时间: 2004
• 期刊: Archives of biochemistry and biophysics.
• 作者: Sivanandam,ArunS;Mohan,Subburaman;Kapur,Sanjay;Kita,Hirohito;Lau,K-HWilliam;Bagi,Gyorgy;Baylink,DavidJ;Qin,Xuezhong
• 通讯作者: Qin,Xuezhong

Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester.

在佛波酯处理的细胞中，研究妊娠相关血浆蛋白 A (PAPP-A) 对 IGF（胰岛素样生长因子）结合蛋白 (IGFBP) 4 蛋白水解的调节。

• DOI: 10.1042/bj20030937
• 发表时间: 2004
• 期刊: The Biochemical journal.
• 作者: Sivanandam,ArunS;Mohan,Subburaman;Kita,Hirohito;Kapur,Sanjay;Chen,Shin-Tai;Linkhart,ThomasA;Bagi,Gyorgy;Baylink,DavidJ;Qin,Xuezhong
• 通讯作者: Qin,Xuezhong