病原體效應蛋白質之結構和功能解析：冠狀病毒之Macro domains及弓形蟲之宿主調控蛋白質PDCD5與Cyclophilin 18

Abstract

病原體劫持了宿主的細胞功能，以便產生有利病原體生存和繁殖的環境。病原微生物，包括分類上屬於真核生物的寄生蟲，原核生物的細菌，甚至病毒都會表達效應蛋白質(Effector protein)。效應蛋白質通常由於通過增加病原體的入侵效率，抑制宿主的免疫系統或啟動病原體的複製，因而對病原體有利。 Macro domain 與涉及DNA 修復、轉錄和免疫反應的轉錄後修飾二磷酸腺苷核糖化（ADP-ribosylation）密切相關。積累至今的證據表示，病毒的macro domain 可透過與二磷酸腺苷核糖（ADP-ribose）分子或其他NAD 的代謝產物相互作用來調節宿主的二磷酸腺苷核糖化修飾。本研究對於MERS-CoV macro domain 與NAD代謝產物結合作用進行調查：透過各種生物物理學的實驗方法在室溫及人體溫度下測量中東呼吸道綜合症冠狀病毒(MERS-CoV)的macro domain 與各種NAD 代謝產物（包括ADP-ribose, NAD, ATP, ADP 和AMP）之間的交互作用。另外我們得到了這些NAD 代謝產物和MERS-CoV macro domain 的複合體晶體結構。藉此我們描述了各種NAD 代謝產物在結構中與蛋白質結合位之間的關係。並且透過核磁共振(NMR)化學位移擾動實驗鑑定了MERS-CoV macro domain 蛋白質結構中，與NAD 代謝物結合相關的關鍵胺基酸。另外，我們利用NMR 實驗方法捕捉MERSCoV macro domain 結合ADP-ribose 及NAD 的動態狀態。在人體溫度環境下，MERS-CoV macro domain 的受質結合區周圍具有彈性的無規則環圈(loop)區域的變化使蛋白質得以結合相較於ADP-ribose 而言具有更大化學結構的NAD。 自2019 年12 月以來，嚴重急性呼吸系統綜合症冠狀病毒2 (SARS-CoV-2)作為新的病原體引起大流行危機。由序列的保守性SARS-CoV-2 被發現表現一個macro domain，且此SARS-CoV-2 macro domain 可能具有影響人類細胞內的ADPribosylation的能力。我們驗證了SARS-CoV-2 macro domain 具有結合ADP-ribose多聚合體的功能，且具有可裂解單個ADP-ribose 的酵素活性。另外我們鑑定了SARS-CoV-2 macro domain 的蛋白質晶體結構。我們的研究提供了針對蛋白質結構，以生物物理和生物化學為基礎，進一步評估SARS-CoV-2 macro domain 與ADPribose結合作用在宿主細胞中的功能，且還可以做為未來設計新藥物的基礎。 弓形蟲病是由原生動物形態的寄生蟲弓形蟲感染所引起的全身性系統性疾病。弓形蟲病對於孕婦和免疫系統不全的人來說是危險的疾病。弓形蟲表現並分泌一種效應蛋白質稱為程序性細胞凋亡蛋白質5（TgPDCD5），會加強宿主細胞的細胞凋亡程度。儘管TgPDCD5 進入宿主細胞的詳細機制仍然未知，但是之前有人提出了一個簡約的論述：TgPDCD5 通過與細胞表面的肝素或硫酸乙醯肝素蛋白聚醣的交互作用，進而誘導胞吞作用而進入宿主細胞。我們的研究為TgPDCD5 的熔球狀特徵提供了確鑿的證據，並且解構了其NMR 水溶液結構，因此建立了TgPDCD5 與硫酸肝素多醣之間的交互作用的結構基礎。另外，在我們的研究中還描述了另一個弓形蟲表現並分泌的效應蛋白質Cyclophilin 18 (TgCyp18)所調節的TgPDCD5 的順反異構變化。相信我們針對TgPDCD5 與TgCyp18 的了解將為未來更近一步研究其功能提供依據。 我們對於MERS-CoV 及SARS-CoV-2 的macro domain 的研究，以及弓形蟲所表現分泌的TgPDCD5 和TgCyp18 的研究，將為病原體的效應蛋白質提供結構與功能方面的新見解。

Pathogens hijack host cellular function to generate an environment which beneficial to their perseverance and propagation. Pathogenic microorganisms, including eukaryotic parasites, prokaryotic bacteria and even viruses expressed effectors are proteins which advantages to the pathogens usually by increasing the invading efficiency, suppressing host immune system, or initiating the replication of pathogens. Macro domains are closely related to ADP-ribosylation, which involves in DNA repair, transcription, and immune response. Accumulated evidence indicated that viral macro domains regulated the host ADP-ribosylation by interacting with the ADP-ribose molecules or other NAD metabolites. Interactions between macro domain from Middle East Respiratory Syndrome coronavirus (MERS-CoV) and NAD metabolites, including ADP-ribose, NAD, ATP, ADP and AMP, were estimated by various biophysical approaches under room temperature and human body temperature. Crystal structures of these metabolites complexed MERS-CoV macro domains were determined. Details in the ligand binding sites of each solved NAD metabolites bound MERS-CoV macro domain were described. And, critical residues of MERS-CoV macro domain for NAD metabolites binding were identified by NMR chemical shift perturbation in solution. Furthermore, dynamic profiles of these interactions were also described. The flexible loops near ligand binding site of MERS-CoV macro domain generated a binding consequence of ligand with larger chemical structure than ADP-ribose, such as NAD, at 35°C. This study provided a systematical elucidation about the ligand binding behavior of MERS-CoV macro domain, especially at the human body temperature. Novel pathogen Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused pandemic crisis since December of 2019. SARS-CoV-2 encodes a conserved macro domain which may reverse cellular ADP-ribosylation. The macro domain of SARS-CoV-2 was examined as a poly-ADPR binding module and possesses mono-ADPR cleavage enzyme activity. Furthermore, crystal structure of SARS-CoV-2 macro domain was determined. Our study provides structural, biophysical and biochemical bases to further evaluate the role of the SARS-CoV-2 macro domain in the host response via ADP-ribose binding but also as a potential target for drug design against new disease. Toxoplasmosis is a systematic disease triggered by infection from protozoan parasite Toxoplasma gondii (T. gondii). This disease can be dangerous for pregnant women and people with compromised immune systems. T. gondii possess an effector protein, programmed cell death protein 5 (TgPDCD5), which involved in host cellular apoptosis enhancement. Although the mechanism of the entry of TgPDCD5 into host cell remained mysterious, a parsimonious suspicion was proposed before which about endocytosis inducing by interaction to extracellular matrixes such as heparin/heparan sulfate proteoglycans. Our research offered solid evidences about the molten globular feature of TgPDCD5 and characterized its solution structure. The structural basis of the interaction between TgPDCD5 and polysaccharide heparin sulfate was revealed. Furthermore, the TgPDCD5 cis/trans-isomerization regulating from another T. gondii effector protein Cyclophilin 18 (TgCyp18) was also described in our study. We believe our pilot elucidation about TgPDCD5 and TgCyp18 will provide the ground of further investigation about their function. Our studies about viral macro domains from MERS-CoV and SARS-CoV-2, furthermore about T. gondii secreted TgPDCD5 and TgCyp18, will provide new insights into the structure and function of these effector proteins from pathogens.