- 學位論文
A-蛋白質激酶1調控痛風發炎經由運輸蛋白訊息傳遞路徑之探討
Investigation of ALPK1 Regulation via MotorProtein Signaling Pathway in Gouty Inflammation
摘要
痛風的特徵於臨床發炎關節中尿酸鹽結晶以急性形式沈積並和A-蛋白質激酶1 (ALPK1) 在尿酸鹽結晶的誘發下與發炎有關。本研究中利用生物資訊、蛋白質體學、細胞模式以及臨床上的調查來釐清在痛風中發炎機制與目標基因ALPK1之間的關聯。我們假設活化階段或單核吞噬細胞系統中成熟的單核細胞對尿酸鹽結晶調節所造成發炎反應，其中ALPK1在生物學功能上參與高基氏體反式網絡結構中了頂端囊泡運輸蛋白質分選的過程，特別是細胞素。因此，ALPK1在痛風的分子機轉上仍然有被研究的必要。我們首度以THP-1細胞為模式利用下拉沈降法配合質譜儀鑑定出ALPK1與其候選蛋白質間的蛋白與蛋白之交互作用，進一步利用全長或截斷蛋白的ALPK1透過免疫沈澱及激酶試驗來進行驗證。發現高Myosin IIA蛋白與ALPK1有高度交互作用並選擇以此來探討其途徑，當ALPK1與Myosin IIA藉由尿酸鹽結晶刺激後發現，並不會增加Myosin IIA蛋白的表現程度但卻會使ALPK1蛋白過度表現。ALPK1鍵結於Myosin IIA的 N端並磷酸化C端使其被活化。Myosin IIA藉著胞吐作用及感測彎曲融合作用來調節再循環內體的囊泡運輸至胞外，小泡的TNF-α會被分泌並誘發發炎反應，另外，我們藉由小分子干擾核糖核酸將ALPK1或Myosin IIA基因敲減後再施以MSU刺激，觀察到在細胞培養液中釋出的TNF-α相較於敲減非目標基因有顯著下降 (p< 0.0001)。這個發現說明在痛風發炎過程中上ALPK1透過磷酸化Myosin IIA調控 TNF-α運輸和分泌，也再次證明MSU活化了ALPK1經Myosin IIA後來誘導 TNF-α的製造。這個結果與另一個發炎途徑活化caspase 1經NALP3 inflammasome誘導 IL-1β的製造不同。在臨床上我們發現未使用藥物之痛風發作患者ALPK1、 Myosin IIA及TNF-α同時高於健康受試者，此結果與建立的細胞模式相呼應。在用藥的痛風患者中TNF-α的下降是是因為秋水仙素抑制了Myosin IIA而不是ALPK1;這個痛風發作的新穎途徑中可能可以藉由秋水仙素抑制來Myosin IIA。此外，ALPK1會和攜鈣素鍵結形成一個複合分子並交互作用於細胞骨架蛋白並且調控胞內小泡運輸分泌的途徑。
並列摘要
Gout is characterized by monosodium urate monohydrate (MSU)-induced arthritis and alpha kinase-1 (ALPK1) is associated with MSU-induced inflammation and gout. In this study, we used bioinformatics, proteomics, cell models, and clinical investigations to clarify the inflammation mechanism targeting ALPK1 in gout. We hypothesized that the state of activation or maturation of monocytes modulates the inflammatory response to MSU crystals in the mononuclear phagocyte system. The biological function of ALPK1 involves in the protein sorting process in the apical vesicle transport at trans-Golgi network, all above cytokine. However, the molecular mechanism of ALPK1 in gout remains to be investigated. We first explored the protein-protein interaction between ALPK1 and candidate proteins in THP-1 cells by pull-down assay with Orbitrap LC/MS/MS protein identification. Then, confirmed by the ALPK1 full-length and truncated forms using co-immunoprecipitation (co-IP) and kinase assay. We found that myosin IIA highly interacted with ALPK1, and was thus selected to explore the pathways. ALPK1 and myosin IIA were stimulated by MSU; myosin IIA did not change, whereas ALPK1 was overexpressed. ALPK1 was bound to myosin IIA at the N-terminal and activated myosin IIA through phosphorylation of the C-terminal. Myosin IIA modulated recycling endosome vesicle trafficking by exocytosis and sensed curvature fusion to extracellular. Vesicular tumor necrosis factor (TNF-α) was secreted, inducing inflammation. Additionally, we have observed through ALPK1/ Myosin IIA gene knockdown by small interfering RNA (siRNA) and, then stimulation with MSU, TNF-α levels were significantly decreased (p< 0.0001) compared with silence non-target genes in culture media. These findings suggested that ALPK1 regulates TNF-α trafficking and secretion through phosphorylation of myosin IIA, accounting for inflammation in gout. MSU crystals may activate ALPK1 and to induce TNF-α production via the myosin IIA. Our results show that this inflammatory pathway distinct from activated caspase 1 and to induce IL-1β production via the NALP3 inflammasome. The clinical investigation findings corresponded with the cell-based MSU stimulation study findings. TNF-α, myosin IIA, and ALPK1 were concurrently higher during gout flares in nonmedicated patients. In medicated gout patients, TNF-α secretion decreased by Myosin IIA, but not by ALPK1, most likely due to colchicine inhibition. This novel pathway could be blocked at the myosin IIA level by colchicine in gout flares. Moreover, the ALPK1－calmodulin complexes may interact with cytoskeleton proteins (F-actin) and mediate the intracellular vesicular transport secretory pathway.