20S蛋白解體與PAN交互作用之分子作用機轉研究

Abstract

在生物體內，泛素-蛋白解體路徑是最主要的蛋白質分解系統。短存活期的蛋白質以及不正常的蛋白質，會經由一連串的酵素反應，進一步被泛素鏈所標示，而多泛素化的蛋白質，隨即會被26S蛋白解體所辨認並且被分解掉。此代謝路徑的主要酵素，26S蛋白解體，是由兩個調節因子包夾一個20S蛋白解體的催化中心所組成。目前，不論在原核生物或真核生物中，都發現20S蛋白解體的存在。但是，20S蛋白解體與調節因子之間的反應機制，以及兩者之間的交互作用為何，到目前為止仍然不甚清楚。 本研究之實驗材料為 Thermoplasma acidophilum之 20S proteasome，其桶狀結 構的外環是由七個相同的 α 次單元所組成，而內環則由七個相同的 β 次單元所組 成。Proteasome activating nucleotidase (PAN) 可結合在 20S蛋白解體的一側或兩 側，形成更具活性之形式。由於PAN是由六個相同的次單元體所組成，不像真核 生物之 19S那麼複雜，因此，對於蛋白解體功能以及其孔道閉合機制之研究，提 供了一個非常有利的研究平台。

為了更進一步了解這兩個分子之間的反應機制究竟為何，本研究針對可能參與交互作用的胺基酸位置進行定位點突變之實驗。目前已成功製備出 Pro17、Lys66、Phe15、Phe22、Arg28、Ala30、Gly34、Lys52、Leu58、Gly80、Leu81、Va l82、Asp84、Arg93、Arg130、Pro131、Gly133、Ala154等突變株，並利用 HPLC分析在有無 PAN參與的情況下，各種帶有不同突變點的蛋白解體，其水解胜肽的反應速率變化情形。 我們發現 Phe15、Phe22 等位置突變之後會大幅提升蛋白解體水解基質的能力，推測可能與蛋白解體基質進出孔道之閉合有關；而 Ala154 則可能與 PAN 之間的交互作用有關。而 Pro17 經過突變之後其水解基質之能力會大幅提升，相反的是，Lys66 經過突變之後會喪失其水解能力，即使與 PAN結合之後也無法提升其活性。最特別的是A30Y突變株，其本身具有極高的基質水解活性，但是與PAN結合之後反而產生了抑制的現象。目前已將此突變株進行結晶，並利用 X-ray 繞射方法解析此 20S A30Y 突變株之結構。另外，我們也發現了一個新的 R130、P131、G133 (RPG) 區塊，推測這個區塊可能參與 α 及 β 次單元體的相互結合，並與調控 α 次單元體基質通道的開啟有關。

The ubiquitin-proteasome system (UPS) is the major proteolytic system in the cytosol and nucleus of all eukaryotic cells. Short-lived proteins as well as abnormal proteins are marked with ubiquitin chains. Polyubiquitinated proteins are subsequently recognized and degraded by the proteasome. The 26S proteasome, the central enzyme of this pathway, comprises the catalytic core particle (20S particle) sandwiched by two regulatory particles (RP or 19S caps). Proteasomes are found in prokaryotes and eukaryotes, but the mechanism and interaction of the 20S proteasome and its regulatory particles are not clear. 20S proteasomes are cylindrical structures with two outer rings each containing seven α subunits and two central rings each containing seven β subunits. In eukaryotes there are seven different α subunits and seven different β subunits, whereas the archaeal 20S proteasome from Thermoplasma acidophilum contains just one type of α subunit and one type of β subunit. The active form of the proteasome in archaea is composed of an ATPase complex such as proteasome activating nucleotidase (PAN) that is bound to one or both ends of the 20S core , which is unlike the composition of eukaryotic proteasome. The simpler PAN-20S complex offers a major advantage to study proteasomal function and its gate opening mechanism.

To further understand the reaction mechanism between these two molecules, we use site-directed mutagenesis to study the sites of the possible amino acids involved. We’ve successfully generated Pro17、Lys66、Phe15、Phe22、Arg28、Ala30、Gly34、Lys52、Leu58、Gly80、Leu81、Va l82、Asp84、Arg93、Arg130、Pro131、Gly133 and Ala154 mutants. Using HPLC, we can analyze the hydrolytic activities of proteasomes with different mutation sites, and compare the difference either with or without the presence of PAN.

We found that after mutation on sites of Phe15 or Phe22, the hydrolytic abilities of proteasome will increase dramatically. This phenomenon might be due to the gate opening for proteasome substrates. On the other hand, the Ala154 mutant might have interaction with PAN. The most special A30Y mutant has the ability to hydrolyze large amounts of substrate, but after binding with PAN, this ability was inhibited. Now we’ve generated crystals of this A30Y mutant, and analyzed the structure of this complex by X-ray diffractions. We also find that RPG domain is very important for the assembly of α and β subunits and for controlling the substrate entry on the α subunit .