探討O連結乙醯葡萄氨糖修飾對於六碳糖激酶的影響以及在阿茲海默症中扮演的角色

Investigation of O-GlcNAcylation on hexokinase 1 in Alzheimer’s disease

蔡叔庭(Shu-Ting Cai)

指導教授 ： 陳韻如

國立臺灣大學/生命科學院/生化科技學系/碩士(2022年)

https://doi.org/10.6342/NTU202203656

摘要

醣化修飾為細胞中最主要的轉譯後修飾，其中將六碳胺基糖生化合成代謝的最終產物胺基糖，經由胺基糖轉移酶催化至蛋白質上絲氨酸或是蘇胺酸位點的醣基化修飾，在細胞中只由一對酵素胺基糖轉移酶和胺基糖分解酶控制整個胺基糖轉譯後修飾，此種修飾通常與蛋白質穩定性、蛋白質分布和蛋白質酵素活性的調節相關。在大腦組織中有許多酵素已知受到胺基糖轉譯後修飾調節，且在阿茲海默症病人的腦組織中發現胺基糖修飾大幅下降。在大腦中主要表現的類型一六碳糖激酶(HK1)分布在粒線體外膜上，參與糖解作用的關鍵步驟將六碳糖催化為磷酸化六碳糖，此一步驟也存在六碳胺基糖生化合成代謝中，和調節細胞中胺基糖水平相關。前人研究當中亦發現當六碳糖激酶(HK1)和粒線體結合能力下降，將會誘發細胞凋亡反應。根據過去實驗室成員的研究成果，已知六碳糖激酶(HK1)存在胺基糖轉譯後修飾，但此修飾對六碳糖胺基糖的生化功能有何影響，仍需進一步探究，為此在本篇研究當中，藉由調整細胞內整體胺基糖水平，在不同水平下觀察六碳糖激酶蛋白質生化功能有何變化。結果發現利用基因方法下降人類神經母腫瘤細胞內的胺基糖轉移酶表現以達到下調細胞胺基糖水平，在此背景下發現六碳糖激酶(HK1)的蛋白質穩定性並不受影響。但上調人類胚胎腎細胞的胺基糖水平卻能夠增加六碳糖激酶(HK1)和粒線體外膜的結合能力，反之下調其胺基糖水平，則能觀測到六碳糖激酶(HK1)和粒線體外膜的結合能力下降。另外，不管在下降或是上調胺基糖水平的背景下測試六碳糖激酶的酵素催化活性並無顯著差異性。由上述結果可以得知細胞內胺基糖轉譯後修飾水平會影響到六碳糖激酶(HK1)細胞內分布定位，但不會影響其蛋白質穩定性以及其酵素催化活性。另外在其他已發表的論文中提到在阿茲海默症的病理模型中，觀察到類澱粉蛋白質(Aβ)大量堆積，並且影響到六碳糖激酶(HK1)與粒線體外膜結合能力，結合本研究內容，我們在人類神經母腫瘤細胞中處理乙型類澱粉蛋白，以觀察其對六碳糖激酶的胺基糖轉譯後修飾或是其他蛋白質生化功能有何影響，在此種背景下進一步探究其在阿茲海默症的病理意義。

關鍵字

醣化修飾 ； 胺基糖糖化修飾 ； 己糖磷酸酶 ； 粒線體能量代謝反應代謝反應 ； 類澱粉蛋白 ； 胺基糖轉移酶 ； 亞細胞定位分布

並列摘要

Glycosylation is considered to be a major post-translational modification. O-GlcNAcylation refers to the addition of N-acetylglucosamine, the end product of Hexosamine Biosynthetic Pathway (HBP), on serine or threonine residues of cytosolic or nuclear proteins through the catalysis by O-GlcNAc transferase (OGT). O-GlcNAc modification is often associated with protein stability, enzyme activity, and subcellular localization. The whole O-GlcNAcylation on proteins is only controlled by O-GlcNAc transferase and O-GlcNAcase in cells. There are many enzymes in brain regulated by O-GlcNAcylation where abnormal O-GlcNAc level has been found in Alzheimer’s disease (AD). Hexokinase 1 (HK1), the brain isoform of hexokinase, is localized at the mitochondria outer membrane. HK1 is the key enzyme in the first step of glycolysis and HBP. It was shown that the detachment of HK1 from mitochondria may cause apoptosis. Previously, we identified HK-1 is O-GlcNAcylated, but the O-GlcNAc effect and sites remains unknown. In this study, using genetic, pharmacological, imaging, and biochemical approaches, I demonstrated that knockdown O-GlcNAc transferase (OGT) in human neuroblastoma BE(2)-C cells caused O-GlcNAc level reduction, but not the protein stability of HK1. Overexpressing OGT in HEK293 cells increased the level of mitochondrial HK1 and the colocalization of HK1 on mitochondria. On the other hand, knockdown OGT in HEK293 cells reduced HK1 level in mitochondrial fraction. As for the enzyme activity of HK1, neither the OGT knockdown group nor OGT overexpression shows a significant difference comparing to the control. These results suggest that the O-GlcNAc level affects the subcellular localization of HK1 but not its stability or enzyme activity in cells. Moreover, some references mentioned that Amyloid-β (Aβ), the pathological hallmark in AD, affects HK1 function. Therefore, I conducted Aβ treatment in BE(2)-C cells to examine the effects on HK1 O-GlcNAylation and function. Together, I provided the effect of O-GlcNAcylation on HK1 and will further investigate the related mechanism in AD.

並列關鍵字

Glycosylation ； O-GlcNAcylation ； Hexokinase 1 ； mitochondrial metabolism ； Amyloid-β ； O-GlcNAc transferase ； subcellular localization

參考文獻

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