巴氏症是一X 染色體變異之罕見疾病，目前此病症多發生於男性幼童。由於病患的Tafazzin 基因缺陷，造成粒線體內心磷脂的生物合成異常，患者多有與粒線體異常之病症。粒線體磷脂水解?(iPLA2)與Tafazzin 的存在，讓心磷脂的合成及水解達到一個平衡的狀態。因此，磷脂水解?(iPLA2)與Tafazzin 皆被認為是治療巴氏症重要的調控?。氫氘交換質譜分析(DXMS)是一項新興的先進科技，目前常用於協助X-RAY 結晶，藥物設計，或是蛋白質結構分析。本研究的目標在於以氫氘交換技術，研究粒線體磷脂水解?(iPLA2) 與Tafazzin 的活化機制。我們首先將建構一套以LC-MS 及MALDI-TOF 雙軌並行的高準度氫氘交換質譜分析系統，研究純化後iPLA2 與Tafazzin 的活化機制。iPLA2 的活化機制包含，ATP 結合而造成的活化、Calmodulin 結合而造成的抑制、Caspase ?切而造成的活化及分子聚合而造成的活化。我們亦將研究Tafazzin 的活化位點，Exon 5 變異的影響，N 端缺陷的結構變化，及重要胺基酸變異所導致的活性缺失。這些機制皆可利用此一氫氘交換質譜分析系統，分析其結構上的改變。最後，我們更將研究這些調控?與一般磷脂膜與粒線體膜的交互作用，以瞭解他們在膜的微環境下的受質結合機制。 Barth syndrome is an X-chromosome linked rare disease, which only occurs in male. The defect of the tafazzin gene causes the abnormality of the cardiolipin biosynthesis in the mitochondria. The patients of Barth syndrome show the mitochondria dysfunction related syndromes. The existence of the calcium-independent phospholipase A2 (iPLA2) and tafazzin creates a balance between the cardiolipin hydrolysis and synthesis in mitochondria. Therefore, iPLA2 and tafazzin are currently considered as the two critical enzymes for drug targets and disease treatment. The Hydrogen/deuterium (H/D) exchange analyzed by mass spectrometry (DXMS) is a useful technology, which has been applied to assist X-ray crystallography, drug design and protein conformation analysis. Our goals here are to use the new DXMS methods to discover the activation mechanisms of mitochondrial iPLA2 and tafazzin. We will setup a parallel DXMS system with both matrix-assisted laser deabsorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography (LC) mass spectrometers to analyze the activation mechanisms iPLA2 and tafazzin. The ATP-binding activation, calmodulin-binding inhibition, caspase-cleavage activation and oligomerization activation will be analyzed by this system. We will also investigate the active site region of tafazzin, the effects on Exon 5 deletion, the conformational changes caused by N-terminal 30-residue truncation, and the activity loss resulted by the critical point mutation. These mechanisms will be investigated by the DXMS system to analyze the structural changes of these enzymes. Lastly, we will further study the interactions between these two enzymes and the mitochondrial membrane to understand their binding mechanisms in the lipid microenvironments.
