Defining the Function(s) of BOG, a pRb Binding Protein

定义 pRb 结合蛋白 BOG 的功能

• 批准号: 6761615
• 负责人: SNORRI S THORGEIRSSON
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6761615
• 关键词: binding proteins; biological signal transduction; carcinogenesis; cell growth regulation; cell proliferation; cell senescence; gene expression; genetically modified animals; genotype; hepatocellular carcinoma; laboratory mouse; neoplasm /cancer genetics; neoplastic growth; neoplastic process; neoplastic transformation; oncogenes; protein structure function; transforming growth factors

Sequence analysis of BOG (RBBP-9) revealed the presence of a putative WXXW binding site for high mobility group protein 1 (HMGB1, amphoterin) HMGB1, spanning amino acids from 23 to 26. HMGB1 is both an abundant component of the cell nucleus and a secreted protein. HMGB1 binds to DNA and bends the double helix, promoting protein assembly on specific DNA targets. HMGB1 secretion occurs through an unknown mechanism, since the protein lacks a leader peptide and does not travel through the endoplasmic reticulum and the Golgi apparatus. Extracellular HMGB1 binds with high affinity to the receptor for advanced glycation end products (RAGE) and promotes outgrowth of cultured cortical neurons, cellular migration and tumor invasion. Additionally HMGB1 was shown to be a potent mediator of inflammation. HMGB1 is a late mediator of endotoxin lethality in mice, and elevated HMGB1 level in septic patients is a poor prognosis marker for survival. HMGB1 can be secreted by macrophages in culture following administration of cytokines like TNF-a and IL-1b and bacterial endotoxin (LPS). The interaction of BOG and HGMB1 was confirmed by co-immunoprecipitation experiments using combinations of anti-HMGB1, anti-BOG and anti-HA antibodies for the detection of HA-tagged BOG. Western blot analysis of the culture medium of RAW 264.7 cells showed that the BOG protein is secreted after exposure to LPS (1?g/ml) or TNF-a (100 mg/ml)in a time-dependent manner. The kinetics of the BOG secretion after exposure to LPS and TNF-a closely mirror the profile of HMG1 release in medium, reaching relatively high levels after 16-18 hours after stimulation. To verify that the release of BOG is not due to leakage of the protein in the medium as a result of cell death, LDH release was assessed during LPS exposure and found to be quantitatively irrelevant if compared to the BOG levels, indicating that BOG secretion is not a passive process consequent to membrane breakage. Like HMGB1, this is an atypical secretion process, since the BOG protein also lacks a leader peptide. The biological importance of the BOG and HMGB1 interaction is currently been pursued in both in vitro and in vivo (ulitizing the BOG -/- mouse model). We have also generated three transgenic lines expressing BOG at different levels and these mice are currently under investigation. Systematic analysis of the animals show consistent abnormalities in BOG transgenic mice as compared to wild-type mice. Among the most significant observations are an early atrophy of the pancreas, consisting of increased incidence of acinar cell apoptosis, increased inter-acinar space with inflammatory infiltrate, loss of acinar architecture and focal proliferation of epithelial-like cells in areas showing apoptotic cells and disorganized acinar architecture. Studies are ongoing aimed at explaining the BOG-induced alterations in pancreas. Histological examinations revealed an increase in cell turnover in the liver of transgenic animals. We are taking two experimental approaches to examine the biological consequences of this observation. The first one is DEN-induced tumorigenesis by i.p. injection of the carcinogen to 15 days animals, and the second one is liver regeneration after 2/3 partial hepatectomy. Both experiments are in progress. Also, we have shown that challenging the kidneys (high BOG expression) in BOG transgenic mice by administration of folic acid (i.p.) following standard procedure results in different kinetics for the injury-reparative response. The results indicate both an acceleration as well as amplification of renal injury based on histological analysis, kidney weight ratio and measurement of blood urea nitrogen. Further studies are necessary to provide molecular mechanisms mediating these differences. These challenging experiments for liver and kidney are being conducted in BOG knock-out animals as well.

BOG（RBBP-9）的序列分析揭示了高迁移率族蛋白1（HMGB 1，白蝶呤）HMGB 1的推定WXXW结合位点的存在，跨越氨基酸23至26。HMGB 1是细胞核中丰富的组分，也是一种分泌蛋白。HMGB 1与DNA结合并弯曲双螺旋，促进蛋白质在特定DNA靶标上的组装。HMGB 1的分泌是通过一种未知的机制发生的，因为该蛋白缺乏前导肽，并且不通过内质网和高尔基体。细胞外HMGB 1与晚期糖基化终产物受体（AGEs）结合，促进培养的皮层神经元生长、细胞迁移和肿瘤侵袭。此外，HMGB 1被证明是一种有效的炎症介质。HMGB 1是小鼠内毒素致死的晚期介质，脓毒症患者中升高的HMGB 1水平是生存不良的预后标志。在给予细胞因子如TNF-α和IL-1b以及细菌内毒素（LPS）后，培养物中的巨噬细胞可以分泌HMGB 1。 BOG和HGMB 1的相互作用通过使用抗HMGB 1、抗BOG和抗HA抗体的组合的免疫共沉淀实验来确认，以检测HA标记的BOG。RAW 264.7细胞培养液的Western印迹分析表明，暴露于LPS（1？g/ml）或TNF-α（100 mg/ml）。暴露于LPS和TNF-α后BOG分泌的动力学密切反映了培养基中HMG 1释放的特征，在刺激后16-18小时达到相对高的水平。为了验证BOG的释放不是由于细胞死亡导致的培养基中蛋白质的泄漏，在LPS暴露期间评估LDH释放，并且发现如果与BOG水平相比，LDH释放在数量上是不相关的，表明BOG分泌不是膜破裂后的被动过程。与HMGB 1一样，这是一种非典型分泌过程，因为BOG蛋白也缺乏前导肽。BOG和HMGB 1相互作用的生物学重要性目前在体外和体内（利用BOG -/-小鼠模型）进行研究。 我们还产生了三个转基因系表达BOG在不同的水平，这些小鼠目前正在调查中。对动物的系统分析显示，与野生型小鼠相比，BOG转基因小鼠中存在一致的异常。最显著的观察结果包括胰腺早期萎缩，包括腺泡细胞凋亡发生率增加、腺泡间隙增加伴炎性浸润、腺泡结构丧失和显示凋亡细胞和腺泡结构紊乱的区域中上皮样细胞局灶性增殖。正在进行旨在解释BOG诱导的胰腺改变的研究。 组织学检查显示，转基因动物肝脏中的细胞更新增加。我们正在采取两种实验方法来研究这一观察结果的生物学后果。第一种是腹腔注射DEN诱发15天动物的肿瘤发生，第二种是2/3部分肝切除后的肝再生。两项实验都在进行中。此外，我们已经表明，通过施用叶酸（i. p.）遵循标准程序导致损伤-修复反应的不同动力学。根据组织学分析、肾重比和血尿素氮测定结果，结果表明肾损伤加速和扩大。进一步的研究是必要的，以提供介导这些差异的分子机制。这些针对肝脏和肾脏的挑战性实验也在BOG敲除动物中进行。