LC3B是p62必不可少的的選擇性自噬但不是基礎(chǔ)自噬PPT課件

1、Biochemical and Biophysical Research Communications446 (2014) 309315阿胡米肯 201101140171 2011級生技2班細胞自噬是一個獨特依賴溶酶體途徑對胞質(zhì)蛋白和細胞器進行降解的一種過程。除了其重要作用通過大量降解提供營養(yǎng)外,這個系統(tǒng)還有能夠選擇性地降解某些蛋白質(zhì),細胞器,入侵的細菌以維持細胞內(nèi)穩(wěn)態(tài)。與酵母中單一Atg8p、哺乳動物中Atg8p 的6個同源染色體對比，LC3和GABARAP組成家庭。作者的研究的目的是確定LC3的單獨作用和GABARAP家庭/本質(zhì)和/或選擇性自噬的基礎(chǔ)。研究結(jié)果表明雖然哺乳動物Atg8同源染

2、色體在功能上對于自噬小體的形成是多余的,選擇性自噬是由特定Atg8同源染色體調(diào)控。巨自噬 (以下稱為自噬)是一種胞內(nèi)降解系統(tǒng)。單膜囊稱為隔離膜或吞噬泡拉長和加固細胞質(zhì)組件包括隨機的整個細胞器。隨后,隔離膜的邊緣相互融合形成一個稱為自噬小體的雙層膜結(jié)構(gòu)。最終,自噬小體通過與溶酶體融合而降解。人的Atg8同源染色體有6個基因密碼，其基因產(chǎn)物可分為兩個亞科;(1)LC3亞科包LC3A, LC3B, LC3C, (2)氨基丁酸受體關(guān)聯(lián)的蛋白質(zhì)(GABARAP)亞科包含GABARAP,GABARAPL1 / GEC-1,GABARAPL2 / GATE-16。雖然以前的研究表明這些蛋白都是共軛與PE,他

3、們似乎有復(fù)雜的無冗余功能在自噬體在膜生物起源和優(yōu)先綁定到適配/目標(biāo)適合于選擇性自噬。這里我們證明LC3 GABARAP家庭都是在HEK293T細胞和Atg8的6個同源染色體可有可無的基礎(chǔ)細胞自噬,LC3B通過自噬對選擇性降解p62負責(zé)。2.12.1、細胞培養(yǎng)和轉(zhuǎn)染、細胞培養(yǎng)和轉(zhuǎn)染HEK293T細胞生長在 (DMEM)含10%胎牛血清(FCS), 5 U/ml青霉素和鏈霉素50 g/的亞融合，在亞融合中 ，使HEK293T細胞轉(zhuǎn)染于控制25 nM 干擾GENOME SMART池子干擾RNA使用DharmaFECT 1轉(zhuǎn)染試劑根據(jù)制造商的協(xié)議(熱科學(xué))。細胞轉(zhuǎn)染48 h后進行分析。不滅的MEF轉(zhuǎn)染

4、了GFP-LC3、GFP-GABARAP GFP-GABARAPL1或GFP-GABARAPL2使用逆轉(zhuǎn)錄病毒載體系統(tǒng),然后用培養(yǎng)基培養(yǎng)的2 g /ml 嘌呤霉素選擇穩(wěn)定遺傳的轉(zhuǎn)化子。2.22.2、 RT-PCR RT-PCRcDNA從1 lg DNase I-treated總RNA使用上標(biāo)第一鏈合成系統(tǒng)(Gibco BRL)和低聚糖(dT)引物。2323、數(shù)字化微滴、數(shù)字化微滴PCRPCR 使用第一鏈規(guī)格互補脫氧核糖核酸合成裝備(羅氏應(yīng)用科學(xué))的互補脫氧核糖核酸合成1 g的總RNA。絕對量化的數(shù)據(jù)顯示。2.42.4。免疫分析。免疫分析 樣本分開使用NuPAGE系統(tǒng)(表達載體)Bis-Tris

5、凝膠12% MOPS-NuPAGE緩沖劑,然后轉(zhuǎn)移到聚乙二烯二氟化物薄膜(PVDF)。 LC3 -或GABARAP p62-staining,細胞被固定和染色anti-LC3B(4價值,MBL)或anti-GABARAP(PM037 MBL)和anti-p62(GP62-C Progen)抗體,分別,如前面描述的那樣。用激光掃描共焦顯微鏡圖像獲得的 2.52.5、長命蛋白質(zhì)降解分析、長命蛋白質(zhì)降解分析 進行的分析是基本上跟之前所述一樣。2.62.6、基于計算機的結(jié)構(gòu)建模、基于計算機的結(jié)構(gòu)建模 GABARAP-LIR和GABARAPL2-LIR復(fù)雜結(jié)構(gòu)模型是基于鼠GABARAP和牛GABARAP

6、L2 / GATE-16分別使用MOE項目.2.72.7、等溫滴定量熱法、等溫滴定量熱法 在PBS ITC實驗進行25 MicroCal-iTC200系統(tǒng)上。在每次運行40 l1.0毫米LIR注入了39倍每隔2分鐘在PBS ITC實驗進行25 MicroCal-iTC200系統(tǒng)上。在每次運行40 l1.0毫米LIR注入了39倍每隔2分鐘從攪拌注射器0.1毫米的樣品室含200 ll LC3或GABARAPL2 / GATE-16。綁定數(shù)據(jù)分析使用5.0和標(biāo)準差起源來自三個獨立的運行。從攪拌注射器0.1毫米的樣品室含200 ll LC3或GABARAPL2 / GATE-16。綁定數(shù)據(jù)分析使用5.

7、0和標(biāo)準差起源來自三個獨立的運行。 (A) RT-PCR analysis of Atg8 homologues in HEK293T cells. (B) Droplet digital PCR analysis of Atg8 homologuesin HEK293T cells. (C) Immunoblot analysis. HEK293T cells were cultured in DMEM containing 10% FCS with or without E64d and pepstatin A (PepA) for 18 h, and then lysateswere c

8、onducted to immunoblot analysis with the indicated antibodies. I: mature form, II: lipidated form.(D) Knockdown analysis. At 48 h after introduction of the indicated siRNA, the cell lysates were analyzed by immunoblotting with the indicated antibodies.(E) Long-lived protein degradation assay. HEK293

9、T cells shown in (D) were labeledwith 14C leucine for 24 h, and degradation of long-lived protein in basal condition was measured. E64d and pepstatin A (E64d + PepA) was added as indicated. Degradationrate of each knockdown cells under normal conditions is indicated as 100%. Data are means SE of tri

10、plicate experiments. P 0.05 and P 0.01.3.1、在HEK293T細胞表達Atg8同源染色體 作者測試了特異性的抗體Atg8同源染色體重組蛋白和驗證他們的特異性,盡管anti-GABARAP和anti-GABARAPL1抗體只表現(xiàn)出適度的降糖藥GABARAPL1和GABARAP,分別(圖S1)。與抗體免疫印跡分析表明,LC3B和GABARAP家族蛋白存在HEK293T。治療HEK293T細胞溶酶體抑制劑、E64d和抑肽素,導(dǎo)致他們的積累PE-conjugated形式以及p62,暗示它們聯(lián)合自噬體溶酶體降解。(A) X-ray crystal structur

11、e of LC3B-LIR (2zjd) and models of the GABARAP-LIR and GABARAPL2/GATE-16-LIR complex from published structures (1eo6 and 1kgt). The molecular surface of each LIR interacting region is displayed with a transparent electrostatic potential(red: negative electrostatic potentials, blue: positive electros

12、tatic potentials). Yellow stick model: LIR peptide consisting of 8 amino acids (337-DDDWTHLS-344) in mouse p62. (B) Representative isothermal calorimetric profiles of LC3B and GABARAPL2 titrated by LIR peptide. Top: raw ITC thermograms, Bottom: fitted binding isotherms. (C) Kinetic data obtained fro

13、m ITC shown in (B). Each bar represents the means SE of three independent experiments. P 0.05, P 0.01. Note the higher affinity of LC3B for LIR compared with GABARAPL2. (D) Precipitation assay. HEK293T cells were transfected with GFP-tagged LC3B, GABARAP or GABARAPL2 together with One-Strep-FLAG tag

14、ged p62 K7A/D69A mutant lacking ability of self-oligomerization 23. At 48 h after transfection, the cell lysates (Total) were subjected to pull down analysis with Strep-Tactin Sepharose, followed by immunoblotting analysis. (Pull-down).3.2、LC3B和GABARAPs對基礎(chǔ)自噬是可有可無的。3.3。 p62 隨LC3家庭在體外相互作用。LC3B. (A) Im

15、munofluorescence analysis. GFP-LC3B, GFP-GABARAP, GFP-GABARAPL1, or GFP-GABARAPL2 was introduced into MEFs, and the MEFs were immunostained with anti-p62 antibody. The right panels show the merged images of GFP (green) and p62 (magenta). Each inset is a magnified image. Scale bars, 10 lm. (B) The pe

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