客户Mol Cell再发新文 阐述自噬调控基因Pacer调控自噬体成熟和脂代谢新机制

前言

近期浙江大学孙启明课题组在Molecular Cell 期刊上发表了题为Pacer is a mediator of mTORC1 and GSK3-TIP60 signaling in regulation of autophagosome maturation and lipid metabolism 的文章，阐释了脊柱生物特有的自噬调控基因Pacer调控自噬体成熟和脂代谢的分子机制。程侠卫博士是论文的第一作者，孙启明研究员是该论文通讯作者。这是该课题组继2017年在Molecular Cell发表Pacer Mediates the Function of Class III PI3K and HOPS Complexes in

Autophagosome Maturation by Engaging Stx17一文后，又一篇关于Pacer功能研究的文章。

背景介绍

自噬是一个进化上保守的过程，涉及溶酶体降解细胞质组分，包括蛋白质，糖原和脂质。自噬功能障碍与多种人类疾病有关。自噬通过感知营养、能量和压力状态在维持细胞和生物体内稳态中发挥重要作用。目前已知的上游调控主要是在自噬体形成的早期步骤起作用；然而，尽管最近在这一领域取得了显著进展，但尚不清楚如何通过环境刺激来调控自噬体成熟。调节自噬的关键上游机制之一是mTOR途径。在营养丰富的条件下，mTOR复合物1（mTORC1）通过磷酸化几种自噬核心元件抑制早期自噬步骤，这些元件包括Unc51样激酶1（ULK1），Atg13，Atg14（L），NRBF2，autophagy / beclin-1 调节因子 1 （Ambra1）和WIPI2。此外，mTORC1还通过磷酸化转录因子EB（TFEB）间接调节自噬流。近年来，随着mTORC1终止自噬并促进自噬溶酶体重组，mTORC1靶向UVRAG抑制自噬体成熟，mTOR通路在自噬后期的关键功能开始显现。  然而，目前尚不清楚mTORC1是否也直接调节了参与自噬晚期所涉及的其他关键因素。

在课题组之前的研究中，作者发现了一种脊椎动物特有的自噬调节器Pacer，并且Pacer促进了自噬体上PI3P的生物发生。此外，Pacer还与STX17协作将HOPS复合体招募到自噬体。本文报道了Pacer调控自噬体成熟和脂代谢的分子机制。

Pacer对肝脏自噬和肝脏内环境平衡至关重要。在自噬诱导条件下，mTORC1介导的Pacer磷酸化被抑制，从而通过GSK3-TIP60途径使Pacer乙酰化。Pacer乙酰化似乎对自溶酶体形成和自噬介导的脂解至关重要。

在这项研究中，研究人员发现Pacer是肝脏自噬和肝脏稳态的调节剂,在营养丰富条件下，Pacer被mTORC1直接磷酸化，进而抑制Pacer与Stx17和HOPS亚基的相互作用，自噬溶酶体的形成；在营养匮乏状态下，Pacer发生去磷酸化，从而可以接收激活的GSK3-TIP60信号而发生乙酰化。乙酰化的Pacer可以更高效地招募HOPS复合物，从而促进自噬溶酶体成熟和肝细胞的脂质代谢。

文章中用到的抗体Pacer及Pacer(Ser157)由华安生物定制生产，另外实验中用到的Beta-Tubulin（M1305-2）也来自华安生物，这些抗体主要用于WB、IHC等。

实验结果图

Pacer对肝脏自噬和脂质平衡的重要作用

(A) Schematic representation of the generation of conditional knockout mice of Pacer. (B) The protein levels of the autophagy markers p62 and LC3 in the livers of four pairs of Pacer f/f and Pacer f/f; Alb-Cre mice for 8 weeks were determined by western blot. (C) Immunohistochemistry showing Pacer protein expression in liver tissues of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 100 mm. 2003, n= 6. (D) Immunofluorescence showing p62 protein levels in liver tissues of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 50 mm. 6003, n = 6.

(E) Immunofluorescence showing LC3 puncta in liver tissues of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 50 mm. 6003 , n = 6. (F) Transmission electron microscopy showing lipid droplets in liver tissues of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 1 mm. 8,3003, n = 6. (G) Oil red O staining showing lipid accumulation in liver tissues of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 100 mm. 2003, n = 6. (H) Periodic Acid-Schiff staining showing hepatic glycogen in liver tissues of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 100 mm. 2003, n = 6. (I) H&E staining showing liver histopathologic changes in liver tissues of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 100 mm. 703, n = 6. (J) Masson staining showing liver fibrosis of 8-week-old Pacer f/f and Pacer f/f; Alb-Cre mice. Scale bars, 100 mm. 2003, n = 6.

在营养丰富的条件下，Pacer 157位点丝氨酸被mTORC1磷酸化

(A) Pacer interacts with mTOR and Raptor. Endogenous Pacer, mTOR, Raptor, and Rictor were immunoprecipitated from HEK293T cells to analyze their interaction by western blot. (B) Alignment of primary sequences of Pacer from various species. Red indicates the identified phosphorylation site of Pacer. (C) The dynamic phosphorylation of Pacer at S157 in response to nutrient status. HEK293T cells were treated with EBSS (Earle’s balanced salt solution) for 15, 30, and 60 min or cultured in complete medium for 2 h after 1 h of starvation. S157 phosphorylation levels were analyzed by phospho-Pacer(S157) antibody. (D) Antibody specificity test. PacerWT-FLAG and PacerS157A-FLAG HEK293T stable cell lines were cultured in complete medium or starved for 1 h. Immunoprecipitation was performed with anti-FLAG beads, and Pacer S157 phosphorylation levels were detected by western blot with phospho-Pacer S157 antibody. (E) Pacer S157 phosphorylation in HEK293T cells that were incubated with mTOR inhibitors (EBSS, Torin1, rapamycin, and KU0063794). (F) Pacer S157 phosphorylation assay in vitro. Endogenous mTOR was purified by immunoprecipitation (IP) from HEK293T cells, and its kinase activity in vitro was analyzed using purified PacerWT-FLAG as substrate at different time points. Torin1 was added to inhibit mTOR activity as a negative control. (G) Autoradiography of Pacer S157 phosphorylation in vitro. Recombinant PacerWT-FLAG and PacerS157A-FLAG proteins that were purified from E. coli were incubated with purified mTOR in kinase buffer containing g-32P-ATP. Pacer phosphorylation (32P-Pacer) was analyzed by autoradiography.

(H) Pacer S157 phosphorylation in Raptor KD HEK293T cells. HEK293T cells were transfected with Raptor shRNA for 72 h. Pacer S157 phosphorylation levels were detected by western blot.

文献来源：

Cheng et al., Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism, Molecular Cell (2018), https://doi.org/10.1016/j.molcel.2018.12.017

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关于华安

杭州华安生物技术有限公司成立于2007年，总部位于中国杭州；2017年在美国波士顿成立全资控股子公司：HUABIO INC. 。华安生物致力于为全球的科研工作者和工业客户提供高品质的抗体试剂及抗体服务。 公司的产品线包括单抗、多抗、二抗、蛋白/多肽、抗体检测用试剂等4000余种。同时，公司也为客户提供定制抗体的开发、纯化和筛选服务。

迄今为止，公司已累计完成超过 3000个定制科研抗体项目，为工业级客户开发IVD诊断用肿瘤标志物系列抗体原料和食品安全检测相关抗体几十余种，为制药公司提供包括PD1和PD-L1热门靶点在内的早期抗体药物筛选十余种。 

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