Ifc之酵素催化能力對神經功能維持及自噬作用調控之研究

Abstract

人類的DEGS1為二羥基神經醯胺去飽和酶(dihydroceramide desaturase)，在神經脂質(sphingolipid)的從頭合成(de novo synthesis)路徑中，負責將二羥基神經醯胺(dihydroceramide)催化為神經醯胺(ceramide)，其蛋白質結構上除了有六個可能的跨膜結構域(transmembrane domains)外，還有三個可能具有酵素催化活性的組胺酸膜體(histidine motifs)。先前的研究指出不同的DEGS1突變會造成神經退化性疾病，而實驗室過去也發現將果蠅的同源基因ifc剔除(knockout)後會造成發育遲緩並在二齡幼蟲階段死亡，若只將果蠅視神經的ifc基因剔除則會造成光依賴性(light-dependent)神經退化，且是由於二羥基神經醯胺堆積所致，顯示Ifc的酵素催化活性對視神經功能維持的重要性。我們將Ifc上對應於病人的位點進行相同突變，並在ifc-KO的情況下全身性過表達這些帶有不同突變的ifc，發現只要突變位置發生在Ifc的組胺酸膜體上即無法拯救果蠅免於死亡。接著我們以西方墨點法測試各組別的蛋白質表現量，結果顯示若將第一組組胺酸膜體或所有組胺酸膜體進行突變，以及N255S的點突變皆會導致Ifc的蛋白質穩定度下降。此外，我們也想測試Ifc的組胺酸模體突變是否會導致二羥基神經醯胺的堆積，結果發現以群組分析(clonal analysis)在果蠅幼蟲眼碟(eye disc)的ifc-KO細胞中過表達帶有第二組或第三組組胺酸模體突變的ifc並不會造成二羥基神經醯胺的訊號有明顯的增加，然而經神經脂質體學分析(sphingolipidomics analysis)則看到在ifc-KO的果蠅幼蟲全身性過表達帶有組胺酸模體突變的ifc確實會導致二羥基神經醯胺/神經醯胺的比值上升。最後我們在果蠅眼睛失去ifc的情況下過表達帶有不同突變的ifc，發現僅有第三組組胺酸膜體的突變無法拯救光依賴性神經退化，此結果顯示了組胺酸膜體對Ifc的酵素催化活性與神經功能維持的重要性。 有研究指出神經醯胺的含量會影響自噬作用(autophagy)的進行，而實驗室過去也發現過表達ifc會促進自噬作用的啟動，然而卻無法順利將自噬小體(autophagosome)進行酸化分解，因此我們想知道過表達ifc是對自噬作用的哪個階段造成影響。結果顯示無論是過表達野生型(wild type)或是帶有第三組組胺酸模體突變的ifc皆會導致自噬小體的標記(marker) GABARAP及自噬小體內作為特殊運送蛋白質(cargo protein)隨自噬作用被分解的ref(2)P表現量上升，而這與我們將syntaxin17的STX17基因減弱(gene knockdown)來抑制自噬小體及溶酶體(lysosome)結合的結果相似，顯示過表達ifc可能是對自噬作用下游的該步驟造成影響，至於為何在過表達野生型或帶有組胺酸模體突變的ifc看到相似的結果，我們推論這是由於突變並沒有完全破壞Ifc的酵素催化活性所致。

Human DEGS1 is a dihydroceramide desaturase that converts dihydroceramide to ceramide in the de novo synthesis pathway. It contains six transmembrane domains and three histidine motifs which are essential for catalytic activity. Previously we have found that loss of ifc, the homolog of DEGS1 in Drosophila, results in the death of larvae at the L2 larval stage, and loss of ifc in Drosophila photoreceptors leads to light-dependent neurodegeneration which is caused by the accumulation of dihydroceramide. According to the studies, different DEGS1 mutations lead to neurodegeneration disorder. To test the impact of these mutations on lethality rescue, we created different ifc point mutations which are the same as the neurodegeneration patients that have mutant DEGS1, and overexpressed in ifc-KO Drosophila. The result showed that only the mutations on histidine motifs cannot rescue ifc-KO lethality. We further used western blot to test the expression level of different ifc mutations, finding that CAT1m, one of the histidine motifs mutant ifc, CATtotalm and N255S affect Ifc protein stability. Also, we found that overexpression of ifc-CAT2m-mCherry or ifc-CAT3m-mCherry did not increase dihydroceramide level in ifc-KO cells of Drosophila eye disc. Nevertheless, sphingolipidomics analysis showed that ifc-CAT2m-mCherry and ifc-CAT3m-mCherry overexpressing increase dihydroceramide/ceramide ratio in ifc-KO Drosophila. Furthermore, we found overexpression of ifc-CAT3m-mCherry cannot rescue light-dependent neurodegeneration in ifc-KO photoreceptor, indicating that histidine motifs play an important role in catalytic activity and neuronal maintenance. Based on research, ceramide level can affect autophagy, and previously we have found that ifc overexpressing can induce autophagy initiation but cannot complete. To further investigate the stage that overexpressed ifc blocks, we used gene knockdown to block different stages of autophagy and observed the signal difference of GABARAP, an autophagosome marker, and ref(2)P, an autophagy cargo protein. The result showed that overexpression of ifc-mCherry or ifc-CAT3m-mCherry increases GABARAP and ref(2)P level. And the phenotype is similar to the syntaxin17 gene knockdown that blocks the fusion of autophagosome and lysosome. As a result, we though that ifc overexpressing can probably block the fusion of autophagosome and lysosome to affect autophagy.