類泛素蛋白酶之基質專一性辨識分子機制研究

已知有超過十種以上的類泛素蛋白質，其胺基酸序列與立體結構皆與泛素有很高的相似性，但是在細胞內確具有不同的的生理功能。類泛素蛋白質中以NEDD8 (Neural Precursor Cell Expressed, Developmentally Down-Regulated 8) 與ubiquitin (Ub) 的序列相似度最高 (有高達80%的胺基酸序列相同)，然而各自的專屬蛋白酶SENP8與USP2卻仍可專一性地針對NEDD8或Ub進行反應。研究指出當蛋白質產生錯誤的NEDD8或Ub修飾時，可能會導致疾病的發生，故探討不同類泛素蛋白質的專一性辨識機制是相當重要的研究課題。本研究以單胞綠藻之bi-ubiquitin CrRUB1 (Chlamydomonas reinhardtii related to ubiquitin 1；其原態為Ub與NEDD8之融合蛋白質) 為基質，利用定位點突變技術將NEDD8與Ub相對應的51及72號胺基酸互換並進行活性分析，發現51號及72號胺基酸分別為SENP8辨識其基質的主要及次要位置，但是這兩個胺基酸的突變並未影響USP2的活性，顯示USP2與SENP8具有不同的基質辨識機制。分析人類USP2與Ub共結晶之結構發現，Ub第4、12與14號胺基酸可與USP2產生交互作用，而且這三個位置的序列也與NEDD8完全不同，故將人類Ub之4、12及14號的胺基酸突變為人類NEDD8之相對應的序列，發現USP2無法有效地水解這些突變株；而將NEDD8之4、12及14號的胺基酸分別突變為Ub之相對應序列後則可被USP2水解，顯示此三個位點是USP2辨識基質的重要胺基酸序列。此外，活性區的重要胺基酸序列及結構與USP2高度相似之USP21也具有相同之基質辨識機制。然而，Ub之 E1-activating enzyme Ube1仍可作用於NEDD8及本實驗所使用之Ub突變株，顯示並非所有參與泛素化修飾之酵素，都具有分辨Ub與NEDD8的能力。

關鍵字

泛素

並列摘要

There are more than ten kinds of ubiquitin-like proteins (Ubls) have been found. Ubls have high similarity of amino acid sequences and three-dimensional structures with ubiquitin, but perform different physiological functions in cells. NEDD8 (Neural Precursor Cell Expressed, Developmentally Down-Regulated 8) and ubiquitin (Ub) have the highest similarity (up to 80%) among different Ubls, whereas the respective proteases SENP8 and USP2 still can react to NEDD8 or Ub correctly. Studies have shown that when proteins with wrong NEDD8 or Ub modification will possibly lead to disease, so it’s important to explore the specific identification mechanism between NEDD8 and Ub modification systems. In the present study, the fusion protein of Ub and NEDD8, CrRUB1 (Chlamydomonas reinhardtii related to ubiquitin 1), was utilized as the substrate for investigating the catalytic specificity of SENP8. By using the site-directed mutagenesis method for replacing the amino acid residues 51 and 72 of CrNEDD8 with their CrUb equivalents, and vice versa, the experimental results showed that CrNEDD8 with N51E/A72R mutations was not cleaved by SENP8, but CrUb with E51N/R72A mutations was efficiently cleaved by SENP8, implying that residues 51 and 72 are the major determinants for specific recognition by SENP8. However, the mutation of residues 51 and 72 of CrNEDD8 and CrUb didn’t largely influence the activity of USP2, suggesting that USP2 and SENP8 have different substrate identification mechanism. The three-dimensional structure of human USP2 and Ub reveals that residues 4, 12 and 14 of Ub could interact with USP2, and these three residues were not conserved in NEDD8. The experimental results showed that Ub4K/12E/14E/72A mutant was not cleaved by USP2, but N84F/12T/14T/72A mutant was efficiently cleaved by USP2, indicating that these four residues were the important determinants for distinguishing between ubiquitin and NEDD8 by USP2. In addition, USP21 also displays the same substrate recognition mechanism. In contrast, Ub E1-activating enzyme Ube1 can still react to NEDD8 and the Ub4K/12E/14E/72A mutant, suggesting that not all the enzymes involved in ubiquitination pathway have the ability to distinguish Ub and NEDD8.

並列關鍵字

Ubiquitin ； NEDD8 ； USP2 ； SENP8

參考文獻

2 Huang, D. T., Walden, H., Duda, D. & Schulman, B. A. Ubiquitin-like protein activation. Oncogene 23, 1958-1971 (2004).

4 Schulman, B. A. a. H., J.W. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. . Mol. Cell Biol. (2009).

5 Komander, D., Clague, M. J. & Urbe, S. Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10, 550-563 (2009).

6 Reyes-Turcu, F. E., Ventii, K. H. & Wilkinson, K. D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78, 363-397 (2009).

7 Kimura, Y. & Tanaka, K. Regulatory mechanisms involved in the control of ubiquitin homeostasis. J Biochem 147, 793-798 (2010).

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類泛素蛋白酶之基質專一性辨識分子機制研究

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