Abstract
摘要:動態而且可逆的SUMO化修飾作用調控了許多重要的細胞生理路徑,而且是生長與發育所必需的。到目前為止,沒有任何專一性抑制劑被報導可以抑制SUMO修飾系統的酵素作用,並且亦不會影響細胞內其它酵素的正常功能。因此為了更進一步瞭解SUMO修飾系統所參與的生理路徑,著實有必要去研發其專一性的抑制劑,用以進行學術研究或者製備成藥劑來治療疾病。雖然已經有幾個研究報告提出,螢光或生物發光共振能量轉移的體外分析平台是一個可行的方法,但是一個容易操作與價格便宜的活體內分析系統則仍未被開發出來。因此本研究計畫擬發展一個活體內的分析系統,利用螢光共振能量轉移為基礎,在大腸桿菌內建構一個能同時表現SUMO活化酶、SUMO銜接酶、與帶有強化型藍螢光蛋白質(ECFP)或強化型黃螢光蛋白質(EYFP)之SUMO-2的組合式SUMO化系統,用以快速篩選出能抑制SUMO化的專一性抑制劑。由於ECFP-SUMO-2與EYFP-SUMO-2在此系統中會互相接合而形成長鏈,使得ECFP與EYFP可以靠近至適當的距離,因此當ECFP受到405 nm的光刺激時,可以將能量轉移給EYFP,而在530 nm偵測到螢光的產生。所以,若在此大腸桿菌的培養基中加入欲篩選的天然物或合成的化學物質,並經過培養及誘導表現後,若無法偵測到螢光共振能量轉移的產生,則表示此物質具有抑制SUMO化的功效。而更重要的是,本方法並不需要利用層析膠體及管柱來進行任何複雜且耗時的蛋白質純化步驟,也不需要加入任何昂貴的酵素基質液於反應步驟中。因此我們認為這個活體內的分析平台,具備容易操作、價格低廉、但是仍能達成快速篩選出SUMO化專一性抑制劑的目的。
Abstract: Ubiquitin and small ubiquitin-like modifier (SUMO) modification systems are related pathways generally using an E1-E2-E3 enzyme cascade to covalently attach ubiquitin or SUMO to a lysine residue of a target protein respectively. The dynamic and reversible post-translational modification by SUMO to target substrates modulates many important cellular processes and is required for viability and development in all eukaryotes.
To date, there is no any reported inhibitor which can specifically inhibit the enzymatic cascade of the SUMOylation system, and also won’t interrupt the normal functions of other enzymes in cells. In order to gain more insights into the SUMOylation system and further investigate the roles of SUMOylation enzymes in all cellular aspects, it is essential to apply a specific inhibitor in the experimental system for understanding this important regulatory pathway and developing the potential medical cures of related diseases. Although two studies have revealed that high-throughput fluorescence resonance energy transfer (FRET)-based or bioluminescence resonance energy transfer (BRET)-based in vitro assay systems are great toolboxes, an easy-to-access and economic in vivo assay platform is still unveiled. Thus, we would like to develop an in vivo FRET-based assay platform in an Escherichia coli chimeric SUMOylation system composed of four co-expressed recombinant proteins, mouse Aos1/Uba2 (E1 activating enzyme), Xenopus Ubc9 (E2 conjugating enzyme), N-terminally ECFP-conjugated human SUMO-2 and N-terminally EYFP-conjugated human SUMO-2, for the rapid screening of SUMOylation-specific inhibitors. Because ECFP-SUMO-2 and EYFP-SUMO-2 will form polymeric chains and therefore bring ECFP and EYFP to a proximal distance in the chimeric SUMOylation system, FRET can be conducted from ECFP (donor, excitation with light of 405 nm) to EYFP (acceptor, emission measured at 530 nm) accordingly. When the SUMOylation activity was inhibited by a specific inhibitor additive (e.g. natural products or some synthetic chemicals in the present proposal) in the culture medium, the polySUMO chains will not be generated, and then the corresponding FRET will not be observed. More importantly, in the proposed in vivo FRET-based assay platform, it is no need to perform any complicated and time-consuming purification procedure with chromatography resins and columns or carry out any in vitro incubation steps by using expensive BRET substrates.
Taken together, this novel FRET-based screening platform in combination with the state-of-the-art biophysical techniques was designed according to the fundamental research results by studying on the SUMOylation enzyme system. We hope that the present proposal can provide an easy-to-access, economic and highly specific screening platform for pharmacologists and pathologists to exploit the potential medical cures of certain related diseases, and also further shed light on the physiological significance of SUMOylation and deSUMOylation systems for biochemists and cell biologists.
Keyword(s)
類泛素蛋白質SUMO
共振能量轉移
SUMO活化酶
SUMO銜接酶
強化型藍螢光蛋白質
強化型黃螢光蛋白質
small ubiquitin-like modifier (SUMO)
SUMOylation
resonance energy transfer (RET)
SUMO-activating enzyme
SUMO-conjugating enzyme
enhanced cyan fluorescent protein (ECFP)
enhanced yellow fluorescent protein (EYFP)