精胺酸與離胺酸甲基化在NRK-52E細胞中調控Klotho蛋白質表現之角色探討

Modulation of Klotho protein expression by lysine and arginine methylation in NRK-52E cells

陳柏宇(Bo-Yu Chen)

指導教授 ： 林庭慧

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2021年)

https://doi.org/10.6834/csmu202100253

摘要

Indoxyl Sulfate ( IS) 為一種尿毒素，是造成慢性腎臟病之致病因子。在尿毒素處理的大鼠腎臟細胞以及慢性腎臟疾病的實驗動物和患有末期腎臟疾病患者的腎臟組織中，具抗老化功能之Klotho蛋白質表現量下降。表觀遺傳修飾，包括DNA甲基化和蛋白質甲基化，可調節Klotho基因的表達。在我們之前的研究中證明了PRMT6和NF-κB 的相互作用導致NF-κB 上精胺酸甲基化並進入細胞核以抑制Klotho表達。為了確認Klotho蛋白質表現的分子機轉，我們研究了NF-κB 上的離胺酸和精胺酸甲基化是否會影響Klotho蛋白質的表現，以及確認Klotho蛋白質本身是否會受到甲基化的調控而影響其穩定性。
我們使用IS處理大鼠腎臟近端小管上皮細胞 (NRK-52E cells)，觀察到IS可以抑制Klotho蛋白質的表現量，而增加精胺酸甲基轉移酶 (PRMT1、PRMT4、PRMT6) 與離胺酸甲基轉移酶 (SET7/9) 蛋白質的表達。若加入DNA甲基轉移酶抑製劑（5-Aza-2'-dc）、專一性的精胺酸甲基轉移酶抑制劑 ( AMI-1)、第7/9型離胺酸甲基轉移酶抑制劑 (Cyproheptadine，SET7/9 inhibitor)，及使用siRNA方法，專一性阻斷細胞內PRMT1、PRMT4、SET7/9的表達，可使被IS抑制之Klotho蛋白質表達回升。這些結果表明DNA甲基化和蛋白質精氨酸、離胺酸甲基化皆可以調節Klotho蛋白質的表達。使用免疫沉澱實驗，確認NF-κB 與Klotho蛋白質上其精胺酸或離胺酸都可被甲基化。此外，利用免疫螢光染色實驗確認 AMI-1及SET7/9 inhibitor可阻斷IS誘導的NF-κB 進入細胞核。利用NF-κB 抑制劑 (Ammonium pyrrolidinedithiocarbamate，PDTC) 處理，可使IS抑制之Klotho蛋白質表達恢復。另外，使用免疫沉澱實驗，確認Klotho與PRMT4、PRMT6、SET7/9有蛋白質交互作用。進一步利用放線菌酮阻斷實驗證明AMI-1可以提升IS抑制之Klotho蛋白質的穩定度。最後，經由PDTC處理NRK-52E細胞，可以降低IS誘導之PRMT1、PRMT4、PRMT6、SET7/9蛋白質表達。我們的研究結果表明在NRK-52E細胞中，蛋白質甲基化在調節Klotho蛋白質的表達扮演重要角色。

關鍵字

硫酸吲哚酚 ； Klotho ； 第一型蛋白質精胺酸甲基轉移酶 ； 第四型蛋白質精胺酸甲基轉移酶 ； 第六型蛋白質精胺酸甲基轉移酶 ； 第7/9型蛋白質離胺酸甲基轉移酶 ； NF-κB

並列摘要

Indoxyl Sulfate (IS) is one of pathogenic factor that cause chronic kidney disease (CKD). In the renal tissues of CKD animal models and patients with end-stage renal disease, expression of Klotho, a protein with anti-aging function, is down-regulated. Cellular DNA and protein methylation reactions play important roles in modulating gene expression. To investigate the molecular mechanism in modulating Klotho protein expression, whether IS-suppressed Klotho expression through protein methylation-dependent pathway in normal rat kidney epithelial cells (NRK-52E cells) was investigated.
Upregulation of protein arginine methyltransferase (PRMT1, PRMT4, PRMT6) and SET domain containing lysine methyltransferase 7/9 (SET7/9) were observed in IS-treated NRK-52E cells, at a concentration that Klotho protein expression was reduced. Diminishment of Klotho expression by IS was restored by a DNA methyltransferase inhibitor (5-Aza-2'-dc), a specific PRMT inhibitor (AMI-1), a specific SET7/9 inhibitor (Cyproheptadine, SET7/9 inhibitor) or treatment with PRMT1 siRNA, PRMT4 siRNA, and SET7/9 siRNA. These results indicated both DNA methylation and protein arginine/lysine methylation modulated Klotho protein expression. Both NF-κB and Klotho protein were identified as substrates for arginine or lysine methylation in NRK-52E cells as detected by immunoprecipitation. In addition, AMI-1 and SET7/9 inhibitor blocked IS-induced NF-κB nuclear translocation in NRK-52E cells as revealed by immunofluorescence staining. IS-suppressed Klotho protein expression was restored by NF-κB inhibitor (ammonium pyrrolidinedithiocarbamate, PDTC). Interaction of Klotho protein with PRMT4, PRMT6, and SET7/9 was confirmed by immunoprecipitation in NRK-52E cells. Moreover, cycloheximide (CHX) blocking assay was used to analyze the effect of IS and AMI-1 on the protein stability of Klotho. Our data indicated AMI-1 restored IS-suppressed Klotho protein expression through maintaining Klotho protein in a more stable status in NRK-52E cells. Finally, expression of the PRMT1, PRMT4, PRMT6, and SET7/9 was upregulated by IS-treatment and was declined by PDTC. Taken together, our results indicated protein arginine and lysine methylation play important roles in modulating anti-aging Klotho protein expression in NRK-52E cells.

並列關鍵字

Indoxyl sulfate ； Klotho ； PRMT1 ； PRMT4 ； PRMT6 ； SET7/9 ； NF-κB

參考文獻

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