植物蛋白水解酶6與受質特異性之生物物理與結構特性研究

Abstract

近年來，極端氣候造成了全球的糧食危機。由極端氣候所引起的乾旱與洪水嚴重影響了水稻的生長並造成了稻米的產量下降。當洪水突然來襲，稻米以及其他植物在淹水的情況下將面臨多重的壓力，進而對它們的生存造成了極大的威脅。植物的N-end rule pathway與其植物體內的蛋白質穩定性有關，在植物體內有一群屬於第七群乙烯反應因子(Group VII ethylene response factors, ERF-VIIs)的蛋白質會被N-end rule pathway中的特定酵素修飾以及辨識。N-end rule pathway中的蛋白酶主要是辨識位於ERF-VIIs 蛋白N端的特定胺基酸，當ERF-VIIs的N端的特定胺基酸被去穩定化之後會被E3連接酶（E3 ligase）的UBR-區域所辨認並且透過泛素化-蛋白酶體系統(ubiquitin proteasome system, UPS)而被降解。在先前的研究中，屬於Arg/N-end rule的第一種類型的N-degron可以被一個具有約70個胺基酸的UBR-區域所辨識。在人類的UBR1 和 UBR2與胜肽（RIFS）的複合物的結構研究中，科學家發現位於胜肽的N端的精胺酸是決定是否與UBR-區域結合的重要因子，此外第二個胺基酸（異亮胺酸）主要是調控UBR-區域與胜肽的結合親和力。植物的N-end rule pathway和第七群乙烯反應因子被認為對於氧氣的感應發揮了重要的作用，因此可以幫助植物對抗淹水逆境。位於ERF-VIIs的N端具有一段為MCGG的保守性序列，此序列會被甲硫胺酸胺肽酶（methionine aminopeptidase, MAP）切掉第一個甲硫胺酸而露出半胱胺酸。當在有氧的環境下，被曝露出來的半胱胺酸會被植物半胱胺酸氧化酶（plant cysteine oxidases, PCOs）氧化，此時被氧化的半胱胺酸是具有負電荷的半胱胺酸亞磺酸（cysteine sulfinic acid），然後接著被精胺酰轉移酶（arginyl transferase）精胺酰化（arginylation）。當ERF-VIIs N端的MCGG透過N-end rule pathway中的蛋白酶而修飾後，這個以Arg-Cysoxo-Gly-Gly為開頭的ERF-VIIs將會被PROTEOLYSIS 6 (PRT6)所辨識而被降解。根據電腦模擬以及序列比對分析，在PRT6的UBR-區域中有兩個獨特且具高度保守性的精胺酸（Arg15 and Arg17）位於與其底物結合的位置。我們認爲帶有正電荷的精胺酸可能與帶有負電荷的半胱胺酸亞磺酸形成鹽橋而產生靜電作用力。根據蛋白結構的電腦模擬數據分析，Arg 15比較靠近與其底物結合的位置，因此在本論文中我們推測在PRT6-UBR-區域中的Arg15可能對於氧化態的N-degron的辨識很重要。 為了驗證我們的假設，我製備了15N-labeled或13C, 15N-labeled的PRT6-UBR-區域的野生種（WT）和突變種 （R15A mutant）的蛋白樣品與透過胜肽合成實驗室合成了模擬ERF-VIIs 的N端的R Coxo GG的合成肽（RDGGAIISDF）。透過核磁共振 （NMR）的實驗來驗證PRT6 的UBR-區域對於RDGG peptide的交互作用。令我們驚訝的是，Arg15的突變造成PRT6-UBR-區域的結構上的改變，而且R15A突變種的結構穩定性降低。但值得注意的是，透過核磁共振 （NMR）的實驗，我們已證明PRT6-UBR-區域的野生種（WT）會與此合成肽（RDGGAIISDF）有交互作用，所以這表示核磁共振 （NMR）的實驗策略將成為一個很好的平台去進行我們的研究。因此我們將繼續透過核磁共振 （NMR）的實驗去探討另一個在PRT6 的UBR-區域中也是具有高度保守性以及獨特性的Arg17對於RDGG peptide的交互作用。此外，我們也更進一步藉由三維的核磁共振實驗去解析PRT6 的UBR-區域與RDGG peptide之間的作用力。目前我們已經從三維的核磁共振實驗收集完所需的的數據以進行更進一步的結構及生物物理方面的研究。

Recently, the extreme climate conditions results in the global food crisis. The drought and flood caused by extreme climate result in great loss of rice growth. The rice and other plants face multiple stresses under submergence because of sudden flood attacks, causing a big threat for the survival of rice and other plants. Interestingly, there is a strategy adopted by the plants that will aid the plants to overcome the flooding. The N-end rule pathway is a targeted proteolysis related to in vivo protein stability based on the N-terminal degron. In this pathway, the destabilizing N-terminal residues of targeted proteins would be recognized by the E3 ligase followed by degradation via ubiquitin proteasome system (UPS). The type-I N-degron of Arg/N-end rule pathway can be recognized by UBR-box domain which possesses a ~70 amino acid zinc finger-like fold. The structural studies of human UBR1 and UBR2 in complexed with N-end rule peptide namely Arginine-Isoleucine-Phenylalanine-Serine (RIFS) used by previous research groups revealed that the first Arginine is decisive for binding recognition and second residue Isoleucine further increase the binding affinity. In plants, N-end rule pathway and a group of transcription factors, ERF-VIIs are believed to play important roles in oxygen sensing. The N-terminal MCGG of ERF-VIIs are modified by methionine aminopeptidase, resulting in exposure of second cysteine. In the presence of oxygen, the exposed cysteine is oxidized by plant cysteine oxidases (PCOs) which form negatively charged cysteine sulfinic acid and then arginylated by arginyl transferase. This unique Arg-Cysoxo-Gly-Gly contains a positive charge and a negative charge and is specifically recognized by plant PROTEOLYSIS 6 (PRT6), an E3 ligase for UPS. Based on computational modeling and sequence analysis, two unique arginines (Arg 15 and Arg 17) of PRT6-UBR domain present in the substrate binding site. In addition, comparing to Arg15, Arg17 is slightly far from the substrate binding site. These two arginines locate near the position 2 of the peptide binding groove; however, the Arg15 is not found in human UBR1/2 and the Arg17 is not found in yeast UBR1. Considering that the arginine may form a salt bridge with negatively charged cysteine sulfunic acid and is conserved among PRT6, we supposed that a conserved arginine (Arg15) in plant PRT6 may be critical for oxidized N-degron recognition. To support our hypothesis, recombinant 15N or 13C, 15N-labelled wild-type and R15A mutant of PRT6-UBR domain were produced and the oxidized N-degron mimic peptide (RDGG) was synthesized for STD-NMR studies to show the conserved Arg is critically involved in cysteine sulfinic acid recognition. To our surprise, the structure of R15A mutant of OsPRT6-UBR domain shows very low stability and its conformation is different from WT OsPRT6-UBR domain. The 2D 1H, 15N HSQC has shown that the WT OsPRT6-UBR domain can interact with peptide mimic (RDGGAIISDF), thus, the NMR experiment is a good platform to understand whether mutant of OsPRT6-UBR domain interacts with peptide mimic (RDGGAIISDF). Therefore, we are creating the R17A mutant which is another conserved and unique residue in plants to study the recognition of OsPRT6-UBR domain with its substrate by NMR experiments. In the future, we will also try to determine the structure of OsPRT6-UBR domain with peptide (RDGGAIISDF) to study the interaction of OsPRT6-UBR domain with its substrates.