多泛素修飾蛋白質純化系統之建立與氧化逆境影響細胞內泛素化修飾作用之探討

Abstract

泛素及類泛素家族為一群真核生物中保留性很高的蛋白質，在生物體的轉錄後修飾中扮演了很重要的角色。泛素前驅物透過去泛素酵素水解其 C 端而顯露出末端之 Gly 後，藉由連續的酵素作用機制與目標蛋白質的 Lys 進行連接。首先 E1 泛素活化酶以耗 ATP 的方式活化泛素，再將泛素移交給 E2 泛素銜接酶，然後藉由 E3 泛素黏合酶將泛素轉移到受質蛋白質上。泛素對於蛋白質的修飾可以是單一泛素化或是以多泛素鏈的形式存在。泛素化修飾系統參與了許多重要的細胞途徑，而泛素蛋白解體系統的失調會造成許多疾病的產生，可見泛素化系統在生物體內所具有的重要性。 生物體在行有氧代謝的過程中會持續的產生活性氧物質 (ROS, reactive oxygen species)，活性氧物質可以作為胞內的二級訊息傳遞者，參與了細胞的訊息傳遞途徑，但是當生物體不正常產生大量活性氧物質而無法正常代謝，或使細胞處於氧化逆境時，會造成核酸斷裂、脂質氧化或是蛋白質受損的情形。由於氧化的受損蛋白質會失去功能或活性，並堆積產生聚合物而造成細胞死亡。因此，細胞選擇性的重新折疊受損蛋白質，或是透過 26S 蛋白解體降解氧化蛋白質以降低對細胞的傷害，是細胞抵抗氧化逆境的保護機制。 為了了解氧化逆境與泛素系統之關係，本論文建立了一個純化多泛素修飾蛋白質的系統，配合蛋白質體學研究，希望可以鑑定出受氧化逆境影響之泛素化蛋白質，並了解這兩者之間的關係。此純化系統藉由 S5a 這個 26S 蛋白解體上的一個多泛素結合次單元體辨認多泛素化受質，再利用去泛素酵素 USP2-core 水解泛素鏈，可得到未受泛素化修飾的受質蛋白質。結果顯示在過氧化氫處理下，293T 細胞內多泛素修飾蛋白質有下降的現象；而 HeLa 細胞則會受到 NO 前驅物 SNP (sodium nitroprusside) 之影響提高多泛素化修飾現象。藉由所建立的純化系統，並利用二維電泳分析，可偵測 293T 細胞中高分子量的泛素複合體受到氧化逆境之後的差異性，但由於 HeLa 細胞對 SNP 的反應不穩定，而無法藉由此系統得到受 SNP 影響的泛素化受質。本研究所建立的多泛素修飾蛋白質純化系統再現性極佳，相信也能多方面運用於泛素蛋白解體的相關研究上。

Ubiquitin and ubiquitin-like family are highly reserved in eukaryotes and play an important role in biological posttranslational modifications. Precursor ubiquitins are processed by deubiquitinating enzymes to expose a C-terminal glycine, and bind with lysine residues on target proteins by successive enzymatic reactions. First, ubiquitin is activated with ATP by E1, the ubiquitin activating enzyme. Ubiquitin is then transferred to E2, the ubiquitin conjugating enzyme, and ligated to a substrate protein by E3, the ubiquitin ligase. Proteins modified by ubiquitins can be either mono- or polyubiquitinated. Since the ubiquitin system is involved in many cellular processes, dysregulation of the ubiquitin-proteasome system leads to diseases, implicating its key role in biological organisms. As a byproduct of aerobic metabolism, reactive oxygen species (ROS) are constantly generated during the lifetime of biological organisms. Reactive oxygen species act as a second messenger in the cellular signal transduction pathway. Cells experience oxidative stress when ROS cannot be removed normally. Under these conditions DNA damage, lipid peroxidation, and protein oxidation take place. These oxidized protein derivatives lose functions and activities, tend to form aggregates. The accumulation of these aggregates may lead to cell death. To prevent this, such oxidatively modified proteins are either selectively refolded or degraded by the 26S proteasome. To study the relationship between oxidative stress and the ubiquitination system, we established a purification system of polyubiquitinated proteins. Coupled with proteomics study, we hope to realize the relationship by identifying ubiquitinated substrates affected by oxidative stress. This purification system utilizes S5a, a polyubiquitinated subunit on the 26S proteasome, to recognize polyubiquitinated substrates and the deubiquiting enzyme USP2-core to hydrolyze ubiquitin chains to obtain target substrate proteins without ubiquitination. The results have shown that, when 293T cells were treated with H2O2, the level of polyubiquitinated proteins decreased while in HeLa cells, treatment with SNP (sodium nitroprusside), precursor of NO, increased the level of polyubiquitination. Together with the purification system and 2 dimensional SDS-PAGE analysis, we can observe the difference in high molecular weight ubiquitin conjugates before and after oxidative stress. Unfortunately, we cannot purify the ubiquitinated substrate affected by SNP with our purification system because the response of HeLa cells to SNP is not stable. The polyubiquitinated protein purification system we created has high reproducibility. Therefore, it can also be useful in studies related to the ubiquitin-proteasome system.