Dataset Information

A natural small molecule alleviates liver fibrosis by targeting apolipoprotein L2 (APOL2)-mediated endoplasmic reticulum (ER) stress

ABSTRACT: To investigate the functional role of APOL2 in liver fibrosis, we performed an unbiased immunoprecipitation-mass spectrometry (IP-MS) analysis for APOL2 in the TGF-β1-stimulated LX-2 cell lysates.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Lu Gan

LAB HEAD: Sheng Yin

PROVIDER: PXD047815 | Pride | 2024-08-13

REPOSITORIES: pride

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A natural small molecule alleviates liver fibrosis by targeting apolipoprotein L2.

Gan Lu L Jiang Qiwei Q Huang Dong D Wu Xueji X Zhu Xinying X Wang Lei L Xie Wei W Huang Jialuo J Fan Runzhu R Jing Yihang Y Tang Guihua G Li Xiang David XD Guo Jianping J Yin Sheng S

Nature chemical biology 20240805 1

Liver fibrosis is an urgent clinical problem without effective therapies. Here we conducted a high-content screening on a natural Euphorbiaceae diterpenoid library to identify a potent anti-liver fibrosis lead, 12-deoxyphorbol 13-palmitate (DP). Leveraging a photo-affinity labeling approach, apolipoprotein L2 (APOL2), an endoplasmic reticulum (ER)-rich protein, was identified as the direct target of DP. Mechanistically, APOL2 is induced in activated hepatic stellate cells upon transforming growt ...[more]

PMID: 39103634

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Project description:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.

			Action	DRS
	APOL2-1.raw	Raw
	APOL2-2.raw	Raw
	APOL2-3.raw	Raw
	APOL2VSIgG-1.msf	Msf
	APOL2VSIgG-2.msf	Msf