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

BOG （RBBP-9）序列分析显示，存在一个推测的高迁移率组蛋白1 （HMGB1，两性素）HMGB1的WXXW结合位点，横跨23 ~ 26个氨基酸。HMGB1是细胞核中丰富的成分，也是一种分泌蛋白。HMGB1与DNA结合并弯曲双螺旋，促进蛋白质在特定DNA目标上的组装。HMGB1的分泌机制尚不清楚，因为该蛋白缺乏先导肽，不能通过内质网和高尔基体。细胞外HMGB1以高亲和力结合晚期糖基化终产物（RAGE）受体，促进培养皮层神经元的生长、细胞迁移和肿瘤侵袭。此外，HMGB1被证明是一种有效的炎症介质。HMGB1是小鼠内毒素致死的晚期介质，脓毒症患者HMGB1水平升高是预后不良的生存标志。HMGB1可由培养的巨噬细胞在给予TNF-a、IL-1b等细胞因子和细菌内毒素（LPS）后分泌。

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.