KLHL20結構域蛋白質之結構及功能研究

Structural and Functional Study of KLHL20 Domain Proteins

葉敏琪(Min-Chi Yeh)

指導教授 ： 何孟樵

國立臺灣大學/生命科學院/生化科學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602778

摘要

由KLHL20所參與的泛素化作用已經被發現會跟腫瘤增生跟細胞自噬終止作用有極大的關係。KLHL20是一個E3連接酶，會去抑制PML和UKL1，PML是一個可以抑制腫瘤的蛋白，而ULK1是參與在細胞自噬的蛋白。所以KLHL20在PML跟ULK1的作用機制中扮演一個重要的角色。當PML的Ser518被磷酸化且Pro519被異構化時就會被KLHL20所辨識，被磷酸化的ULK1已知會被KLHL20所認，因此KLHL20被認為會用不同種的方式辨識其他蛋白。但是目前KLHL20的被辨識機制並不是很清楚，我們合作的實驗室已經發現KLHL20會跟不同種類的蛋白作用產生不同的路徑，目前已知的是KLHL20會跟Cul3和Roc1作用形成複合體再跟其他蛋白作用。但是像是KLHL20的受質PML也是一個蛋白載體，因此用Pull-down的方式很難去找出其他KLHL20的受質蛋白。所以我們的目的是要去找出其他會跟KLHL20作用的蛋白，利用從可能的受質蛋白序列合成胜肽，再將KLHL20直接跟胜肽做反應，找出特定的序列，再從中找到其他受質蛋白。再進一步的解出蛋白和胜肽的複合體結構跟發展治療癌症藥物。

關鍵字

KLHL20 ； 腫瘤增生 ； 細胞自噬終止作用 ； OBOC ； 泛素化

並列摘要

KLHL20-mediated ubiquitination is highly related to tumor progression and autophagy termination. KLHL20, an E3 ligase down regulates PML and ULK1, which are tumor suppressor and autophagy proteins, respectively. Hence, KLHL20 is a key substrate adaptor for both PML and ULK1 pathways. The recognition of PML by KLHL20 requires both serine phosphorylation at Ser518 and the isomerization at Pro519. ULK1 autophosphorylation is required for KLHL20 recruitment but the isomerization is not identified. These findings indicate that KLHL20 can interact with protein substrates in different fashions. However, the molecular recognition mechanism of KLHL20 is not yet clear. Our collaborator has found that KLHL20 may be involved in different cellular functions by interacting with other protein substrates. KLHL20 is known to interact with Cullin3 (Cul3) and Roc1 and often forms complexes with other proteins. Some of KLHL20 substrates such as PML also interact with other proteins. Hence, it is very difficult to identify KLHL20 substrates by conventional pull-down assays. Currently, we plan to identify KLHL20 by biophysical approaches. We aim to develop direct binding assays to examine if the peptide sequences from the potential substrates can interact with KLHL20 directly. We also hope to identify the critical sequence for KLHL20 binding using one bead-one compound combinatorial chemistry (OBOC) approach. Our results can facilitate KLHL20 substrate identification. Our ultimate goals are to determine the structures of KLHL20-substrate complexes and develop the potential drugs for cancer therapy.

並列關鍵字

KLHL20 ； Tumor progression ； Autophagy termination ； OBOC ； protein-protein interactions

參考文獻

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