Abstract
摘要:Poly ADP-ribosylation是相當重要的後轉譯修飾,涉及到許多細胞的活動,訊息傳遞以及遭受逆境的反應調控,例如單股去氧核糖核酸斷裂。Poly ADP-ribose的修飾循環是相當重要的細胞程序與調節,長鏈分支狀的poly ADP-ribose (PAR)是由PAR聚合酶以NAD為原料所生成,而一種序列含有macro domain的酵素,PARG則為斷裂這類長鏈分子的主要酵素。PARG是相當具有保守性,從細菌到人類都含有這樣的酵素。有趣的是,含有macro domain的酵素相當的多樣性,並催化著各種ADP-ribose相關的代謝物。此外,含有macro domain的蛋白質也具有辨識poly ADP-ribose的功能。
在這個計畫,我們目標在於研究DrPARG和CoV冠狀病毒含有macro domain的蛋白。因為我們的初步結果顯示DrPARG有可能是未曾被報導過的細菌型外切PARG酵素,而CoV的病毒蛋白研究有助於我們對抗此類新興的傳染疾病。本三年計畫旨在以系統性地利用野生型、各突變株之具有macro domain之微生物PARG與病毒蛋白配合添加各受質等方式,對於酵素的催化機制與基質辨識進行物理化學特性探索與結構闡述。此研究將延續我們建立的系統與結果提出了四個目標:
1) 解析不同含有macro domain之酵素與蛋白的原子層級立體結構與催化活性中心◦ 2) 瞭解macro domain的蛋白折疊與動態及其它物理化學特性◦ 3) 分析及比較macro domain蛋白家族與其受質結合區的構型與胺基酸組成喜好差異◦ 4) 解構含macro domain酵素的保守胺基酸於催化機制之作用與重要性◦
為達成這些目標,我們已成功生產數種含有macro domain的酵素與蛋白並獲得部分蛋白質晶體。我們亦已構築或取得其它macro domain的表達載體及數個突變株,在取得高純度的蛋白樣本後,除了解析蛋白自身的結構外,也將從事酵素與受質類似物或抑制劑形成之複合體的結構解析◦而如初步資料所示,配合數種物理化學分析方法,如核磁共振光譜、量熱儀等測量,將可釐清酵素催化機制與基質辨識。此外目前並無macro domain相關的蛋白折疊與動態等物理化學特性分析,我們將首度進行此項研究並冀望能提供充分資訊。基於這些豐富的初期結果與確切目標,上述之各個研究主題應能順利達成◦
Abstract: Poly ADP-ribosylation is an important post-translational modification (PTM) which is involved in various cellular processes, mostly in cellular signaling and responses to cellular stress such as DNA single strand breaks (SSB). Poly ADP-ribose turnover is an important process in cellular functions. Long branched poly ADP-ribose (PAR) polymers are formed on target proteins by PAR polymerase (PARP) utilizing nicotinamide adenine dinucleotide (NAD+) as a substrate. A macro domain-containing enzyme, PAR glycohydrolase (PARG) is the major enzyme catalyzing the breakdown of poly ADP-ribose chains. PARG consist a conserved family of enzymes from bacteria to human. Interesting, macro domain-containing enzymes have diverse catalytic activities for ADP-ribose metabolite; while macro domain-containing protein could recognize the PAR molecules.
In this proposal, we choose two macro domain-containing proteins, PARGs and viral proteins as our research targets. Since our preliminary studies of DrPARG indicate it could be an exo-PARG, which has not been report yet. While X-domain and SUD domain from SARS-CoV EMC are little known and may be a target for against the emerging infectious disease. Catalytic mechanism and substrate recognition of microbial PARG and viral proteins would be systematically approaching with at least wild-type, mutants as well as addition of ligands via physicochemical characterization and structural elucidation. Therefore, we would like to engage in the following studies:
1) Obtain atomic resolution pictures of overall structural architecture of macro domain-containing viral proteins and enzymes. 2) Determine the physicochemical properties, folding and protein dynamics of macro domain-containing protein. 3) Compare the ligand-recognition differences among these macro domain-containing proteins. 4) Decipher the roles of conserved residues in the catalytic mechanisms of macro domain-containing enzymes.
Towards these goals, we have constructed/obtained expression plasmids for expressing soluble forms of various macro domain-containing protein, specifically DrPARG and CoVEMC X and SUD domains, and several protein crystals’ conditions have been screened out. After purified proteins are available, various binary complexes formed between enzyme(s) and substrate analog(s)/product(s) as well regulatory protein(s) will be used for crystallization. Moreover, a series of mutant enzymes with altered catalytic behavior has been identified and their structures will also be examined by various physicochemical methods, such as NMR, ITC. In addition, there is no any report regarding to protein folding, dynamics and biophysical chemistry studies of macro domain protein family, we would like to provide the important imformation. On the basis of our solid preliminary results and the certain approaches, it is expected that significant progress toward understanding the structure/functional relationship of PAPG enzymes family will be achieved.
Keyword(s)
macro domain蛋白家族
蛋白質結晶學
核磁共振光譜技術
生物物理化學
ADP-ribose修飾
macro domain protein family
protein crystallography
NMR technique
biophysical chemistry
ADP-ribosylation