人類胸腺嘧啶激酶及胸苷酸激酶降解機制之探討

Degradation of human thymidine kinase 1 and thymidylate kinase: involvement of the anaphase-promoting complex/cyclosome-mediated proteolysis

柯博元(Po-Yuan Ke)

指導教授 ： 張智芬

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00014

摘要

細胞內適量的胸苷三磷酸(dTTP) pool對於DNA複製以及修補的正確性是相當重要的，細胞中主要有二條路徑可以提供胸苷三磷酸(dTTP)的合成，分別是de novo pathway以及salvage pathway。在de no pathway這一條合成的路徑中，胸苷酸形成酶(thymidylate synthase)在將dUMP轉變成胸苷酸(dTMP)的反應中扮演著速率決定步驟的角色；然而，在另外一條salvage pathway上，胸腺嘧啶激酶(thymidine kinase)負責將腺苷三磷酸(ATP)的5’末端的磷酸根(phosphate group)轉移至胸腺嘧啶(thymidine)，進而形成胸苷酸(dTMP)，隨後，無論是經由de novo或是salvage形成的胸苷酸(dTMP)都必須經由胸苷酸激酶(thymidylate kinase)進一步的磷酸化形成胸苷二磷酸(dTDP)，再透過核苷二磷酸激酶(NDK)的磷酸化最後完成胸苷三磷酸(dTTP)的合成。為了能配合細胞週期中DNA合成的時間，細胞中胸苷三磷酸(dTTP)的合成必須隨著細胞週期的進行而被嚴密調控著。當細胞週期即將進入到S phase時，胸苷三磷酸(dTTP) pool會比在G0 phase增加了近20倍，為了可以快速地形成如此大量的胸苷三磷酸(dTTP)，胸苷酸形成酶(thymidylate synthase)、胸腺嘧啶激酶(thymidine kinase)以及胸苷酸激酶(thymidylate kinase)必須經由轉錄作用的調控(transcriptional control)，而使得表現量增加，得以應付大量合成胸苷三磷酸(dTTP)時所需要的酵素。隨著DNA複製的完成，胸苷三磷酸(dTTP)不再被需要時，合成的反應必須被抑制；然而，至今對於細胞如何調降胸苷三磷酸(dTTP)合成的研究卻是付之闕如。

細胞分裂後期促進複合體(Anaphase-promoting complex/cyclosome, APC/C)所主導的蛋白質降解機制對於染色體分裂、脫離細胞分裂以及進入G1 phase是相當重要的。有二個活化子，包含Cdc20及Cdh1可以協助APC/C辨識即將要被降解的蛋白質；APC/C-Cdc20主要透過辨認蛋白質上的D-box (RXXL)而APC/C-Cdh1則辨認D-box (RXXL)和KEN-box (KEN)。在本論文中，我將研究重心著重於探討蛋白質降解機制對於細胞中胸苷三磷酸(dTTP) pool濃度的調節作用，我證明了APC/C所主導的蛋白質降解作用是經由分解胸腺嘧啶激酶(thymidine kinase)以及胸苷酸激酶(thymidylate kinase)，而在細胞中胸苷三磷酸(dTTP) pool的調控上扮演著舉足輕重的角色。

首先，在本論文的第二章，我證明了人類胸腺嘧啶激酶(thymidine kinase)在細胞即將脫離分裂期的時候會經由ubiquitin-proteasome pathway而大量被分解；APC/C-Cdh1在這個降解的過程中是一個必要的速率決定者。這樣經由APC/C-Cdh1所主導的胸腺嘧啶激酶(thymidine kinase)降解是透過Cdh1與一個位於胺基酸序列上C端上的KEN-box交互作用所達成的，藉著in vitro polyubiquitinylation的實驗，我更加證實了這一個降解機制對於胸腺嘧啶激酶(thymidine kinase)穩定性的重要性。

在本論文的第三章，我發現了胸苷酸激酶(thymidylate kinase)也是另一個可以被APC/C分解的蛋白質。藉由in vivo以及in vitro的實驗，我也證明在細胞分裂以及G1 phase的初期，APC/C-Cdc20以及APC/C-Cdh1都會導致胸苷酸激酶(thymidylate kinase)的降解，而這一個過程中是經由辨認胸苷酸激酶(thymidylate kinase) 胺基酸序列上的一個D-box以及另一個KEN-box所完成的。最後，為了能了解增加這二個酵素表現量對於細胞中胸苷三磷酸(dTTP) pool的影響，我在細胞中同時地表現原生型(wild type)的胸腺嘧啶激酶(thymidine kinase)及胸苷酸激酶(thymidylate kinase)，發現了細胞中胸苷三磷酸(dTTP) pool大幅增加了4至5倍;反之，同時表現不分解的突變型(nondegradable mutant)的胸腺嘧啶激酶(thymidine kinase)及胸苷酸激酶(thymidylate kinase)使得細胞中胸苷三磷酸(dTTP) pool更大幅增加至10倍，伴隨著產生嚴重的dNTP pool imbalance，導致細胞生長速率的下降以及延緩細胞週期中S phase到G2/M phase的進行。更重要的是，利用hprt (hypoxanthine-guanine phosphoribosyltransferase)基因自發性的突變率分析，我證明了當細胞同時表達表現不分解的突變型(nondegradable mutant)的胸腺嘧啶激酶(thymidine kinase)及胸苷酸激酶(thymidylate kinase)呈現令人驚訝的突變率上升；然而表達原生型(wild type)的胸腺嘧啶激酶(thymidine kinase)及胸苷酸激酶(thymidylate kinase)並沒有造成任何基因突變率增加的現象。因此，在本篇論文中，我證明了APC/C所主導的蛋白質降解作用在調控細胞中dNTP pool balance中扮演著極為重要的角色，這樣的分子調控制機制對於維持基因體的完整性及穩定性是相當必須的。

關鍵字

胸腺嘧啶激酶胸苷酸激酶蛋白質降解

並列摘要

Proper control of intracellular dTTP pool size is critical for maintenance of high fidelity of DNA replication and DNA repair. Concurrent supply of dTTP relies on functions of both the de novo and salvage pathways. In the de novo pathway, thymidyltae synthase (TS) catalyzes the rate-limiting step of converting dUMP to dTMP. Alternatively, thymidine kinase (TK), the key enzyme in the salvage pathway, catalyzes the reaction transferring the terminal phosphate of ATP to the 5’-hydroxyl group of thymidine to form dTMP. Subsequent phosphorylation of dTMP by thymidylate kinase (TMPK) gives rise to dTDP, which is then converted to dTTP by dNDP kinase (NDK) for DNA synthesis. Intracellular production of dTTP is a highly regulated process, which is coordinated with DNA replication in the cell cycle. As cell approaches S phase, dTTP pool size is increased to 20-fold than that in G0-phase cells. This up-regulation of dTTP can be attributed to cell cycle-dependent expression of TS, TK, and TMPK through transcriptional control. After the completion of DNA replication, dTTP is no longer demanded, the forward synthesis must be inhibited. However, little is known about how the dTTP-synthesis enzymes are down-regulated. Anaphase promoting complex/cyclosome (APC/C)-mediated proteolysis is essential for chromosome segregation, mitotic exit and G1 entry. Two activators, Cdc20 and Cdh1 target substrates for APC/C-mediated proteolysis in mitosis and G1 phase, respectively. APC/C-Cdc20 prefers to recognize the D-box (RXXL), while APC/C-Cdh1 binds to the D-box and KEN-box (KEN). In this thesis, I demonstrate that the APC/C plays an important role in controlling dTTP pool size by targeting degradation of human cytosolic TK (hTK1) and TMPK (hTMPK). In chapter 2, I show that hTK1 is degraded via a ubiquitin-proteasome pathway in mitotic exit phase and that APC/C-Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. A KEN-box sequence located in the C-terminal region of hTK1 is required for its recognition by Cdh1. By in vitro polyubiquitinylation assays, I provide direct evidence that APC/C-Cdh1 is a direct ubiquitin E3 ligase of hTK1 by recognizing its KEN box. In chapter 3, I further identify that hTMPK is another target of APC/C. By in vivo and in vitro experiments, I demonstrate that both APC/C-Cdc20 and APC/C-Cdh1 mediate hTMPK for degradation. APC/C-Cdc20 recognizes hTMPK through D box in M phase whereas APC/C-Cdh1 binds to D- and KEN boxes in early G1 phase. Simultaneous expression of wild type hTK1 and hTMPK leads to a 4-5-fold increase of the dTTP pool without promoting the spontaneous mutation in the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene. In contrast, co-expression of nondegradable hTK1 and hTMPK expands the dTTP pool size ten-fold and induces a drastic dNTP pool imbalance, accompanied by a decrease in growth rate and a delay in SàG2/M transition. Most interestingly, cells co-expressing nondegradable TK1 and TMPK display a striking increase in gene mutation rate. I conclude that down-regulation of dTTP pool size by APC/C pathway during mitosis plays a critical role in keeping a balanced dNTP pool, which is essential for the S phase progression and the maintenance of genome integrity.

並列關鍵字

thymidine kinase ； thymidylate kinase ； proteolysis ； APC/C

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