Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells

Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca2+ concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca2+ stores and influx of extracellular Ca2+, which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca2+ antagonists, including calbindin-D-28k (an intracellular Ca2+ buffering protein) and BAPTA-AM (a cell-permeable Ca2+ chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca2+ concentration and its link with AA-induced cytotoxicity

Evolutionary Studies on Unique Proteins of Oil Bodies

本實驗室過去十餘年於國科會計畫長期支持下選擇芝麻種子油體當模型系統(model system)從事油體蛋白質之研究。油體構造是一團三酸甘油酯包在一層磷脂質(phospholipid)內，此磷脂質層鑲滿豐富的構造蛋白質叫油體膜蛋白(oleosin)及二種微量蛋白質名為油體鈣蛋白(caleosin)與油體固醇蛋白(steroleosin)。被子植物種子油體膜蛋白有兩個同功蛋白(oleosin-H and -L)，油體固醇蛋白也有兩個同功蛋白(isoform A and B)。2007年我們首次從百合花粉中純化到穩定的油體，並找到另一個油體膜蛋白同功蛋白(oleosin-P)。裸子植物松(Pinus koraitusis)、銀杏(Ginkgo biloba)的油體中僅存在一種油體膜蛋白，為oleosin-L；然而蘇鐵(Cycas revoluta)油體並無油體膜蛋白，卻含有分子量約為30 kDa的蛋白質，本研究室初步結果顯示極可能是油體鈣蛋白。本計畫擬研究油體蛋白質的演化關係。第一年擬探討花粉油體中是否含有油體鈣蛋白與油體固醇蛋白，或是其他未知蛋白質，並篩選定序其對應基因。此外，將構築蘇鐵種子形成過程中油體累積時期之cDNA library以供尋找並定序蘇鐵油體蛋白基因。第二年擬篩選定序蘇鐵油體蛋白質對應基因，證實油體鈣蛋白為蘇鐵油體之最大量蛋白質，並對蘇鐵油體做較深入的生化分析。此外，擬找尋比松更高等的裸子植物(例如，麻黃)及較原始的被子植物(例如，八角)的油體，以便瞭解oleosin-H在演化上何時從oleosin-L複製分出。第三年擬篩選定序麻黃或八角油體膜蛋白對應基因，同時檢測蘇鐵、麻黃、八角及演化上更早期的生物(例如，蕨類、藻類)是否有油體固醇蛋白或其他未知蛋白質。期許提供更多實驗數據，推論油體蛋白質的演化關係。In the past decade, our group worked on sesame oil body proteins under the support of NSC grants. Seed oil bodies contain a triacylglycerol matrix surrounded by a layer of phospholipids and unique proteins. Three classes of oil body proteins termed oleosin, caleosin, and steroleosin have been identified in sesame oil bodies. Isoforms were found in oleosin (H and L forms) and in steroleosin (A and B forms). In 2007, we first isolated stable oil bodies from lily pollen and identified a new class of oleosin (P-isoform). Only L-oleosin but not H-oleosin was found in oil bodies from pine and ginkgo seeds. While no oleosin was detected, a putative caleosin of 30 kDa was found in cycas seed oil bodies. In this proposal, we plan to investigate the evolution of oil-body proteins. In the first year, we intend to detect proteins other than oleosin-P in pollen oil bodies and to clone their corresponding genes. In addition, a cDNA library will be constructed with mRNA extracted from maturing cycas seed. In the second year, we intend to clone the corresponding gene for the putative caleosin found in cycas seed oil bodies, and characterize these oil bodies in depth. To know the splitting of oleosin-H from oleosin-L, we schedule to analyze oil bodies and their proteins in Ephedra or Illicium. In the third year, we intend to clone and sequence genes encoding putative oil-body proteins from cycas, Ephedra, Illicium, or other primitive species such as ferm and algue. We expect to provide more experimental evidence for the analyses of evolution in oil-body proteins

Molecular Mechanism of Triacylglycerol Lipase on the Surface of Seed Oil Bodies

本實驗室過去十餘年於國科會計畫長期支持下選擇芝麻種子油體當模型系統(model system)從事油體蛋白質之研究。油體構造是一團三酸甘油酯包在一層磷脂質(phospholipid)內，此磷脂質層鑲滿豐富的構造蛋白質叫油體膜蛋白(oleosin)及二種微量蛋白質名為油體鈣蛋白(caleosin)與油體固醇蛋白(steroleosin)。關於脂肪分解酵素(lipase)如何作用於種子油體表面而順利將三酸甘油酯中的脂肪酸分解出來送至glyoxyosome進行β-oxidation產生能量的機制則尚未被研究，因為分解植物種子油的脂肪分解酵素及其對應基因直到今年才被報導出來 (Eastmond 2006，從阿拉伯芥獲得)。目前實驗室利用已發表的阿拉伯芥脂肪分解酵素從芝麻發芽種子中獲得脂肪分解酵素cDNA基因部分片段。本計劃擬用三年時間研究脂肪分解酵素如何作用於種子油體表面，特別是與油體表面蛋白質之特定交互作用，探討油體膜蛋白、油體鈣蛋白、或油體固醇蛋白是否有擔任脂肪分解酵素「接受器」（lipase receptor）的角色。第一年將完成芝麻發芽種子中脂肪分解酵素cDNA基因全長，並尋找芝麻種子是否有其他同源脂肪分解酵素。也將芝麻脂肪分解酵素於細菌E. coli大量表達，純化後用於製備抗體。第二年將利用抗體偵測脂肪分解酵素於芝麻成熟中與發芽後種子含量的變化，並利用免疫金原子標定技術在電顯下偵察芝麻脂肪分解酵素於種子胞內分佈情形及是否於出現於發芽後油體表面。此外將以抗體協助純化芝麻脂肪分解酵素，提供酵素特性分析。同時嘗試將芝麻脂肪分解酵素於酵母菌表達，以便大量取得有活性之酵素。第三年將測試芝麻脂肪分解酵素作用於純化之芝麻油體及人造芝麻油體，希望利用不同油體蛋白質組成的人造芝麻油體被芝麻脂肪分解酵素作用的情形，來找尋油體膜上的脂肪分解酵素「接受器」。In the past decade, our group worked on sesame oil body proteins under the support of NSC grants. Seed oil bodies contain a triacylglycerol matrix surrounded by a layer of phospholipids and unique proteins. Three classes of oil body proteins termed oleosin, caleosin, and steroleosin have been identified in sesame oil bodies. How lipase works on the surface of seed oil bodies to release fatty acids that are transferred into glyoxysome for β-oxidation has not been addressed. The first clone of lipase responsible for the degradation of seed triacylglycerol was reported this year from Arabidopsis (Eastmond 2006). According to the sequence of Arabidopsis lipase, we have obtained a partial cDNA clone encoding sesame triacylglycerol lipase. In this grant proposal, we intend to study the interaction between sesame lipase and oil body proteins, and thus to identify the lipase 「receptor」 on the surface of oil bodies in the following 3 years. In the first year, the complete sequence of sesame lipase will be obtained and over-expressed in E. coli. The expressed lipase will be purified and subjected to antibody generation. Homologous genes encoding lipase will be cloned from developing and germinating seeds. In the second year, the abundance of lipase in developing and germinating sesame seeds will be analyzed by Western blots. Immunogold detection will be used to track if lipase appears on the surface of oil bodies after germination. Native lipase will be purified and characterized from post-germinative sesame seeds under the assistance of antibody. Recombinant lipase will be produced in yeast to harvest active lipase in a large scale for the following investigation. In the third year, active lipase will be used to digest triacylglycerol in purified sesame oil bodies or artificial sesame oil bodies constituted with different compositions of oil body proteins. Based on these analyses, we expect to identify the lipase receptor on the surface of sesame oil bodies