pSILAC method coupled with two complementary digestion approaches reveals PRPF39 as a new E7070-dependent DCAF15 substrate

doi:10.1016/j.jprot.2019.103545

	pSILAC method coupled with two complementary digestion approaches reveals PRPF39 as a new E7070-dependent DCAF15 substrate
	Jia, Xinglong 2; Pan, Lulu 3,4; Zhu, Mingrui 3,4; Hu, Hao 3; Zhai, Linhui 3; Liu, Jie 1; Hu, Min 1; Liu, Bin 1; Tan, Minjia 3,4
刊名	JOURNAL OF PROTEOMICS
	2020-01-06
卷号	210 页码:11
关键词	E7070 pSILAC LysC-Trypsin LysN-LysArgiNase PRPF39 DCAF15 Degradation
ISSN号	1874-3919
DOI	10.1016/j.jprot.2019.103545
通讯作者	Liu, Bin(liubin3575630@163.com) ; Tan, Minjia(mjtan@simm.ac.cn)
英文摘要	Targeting specific ubiquitin E3 ligase for degradation of disease-driven protein has recently been an important concept for cancer therapy, as exemplified by the case of thalidomide for the treatment of multiple myeloma. E7070, an aryl sulfonamide drug, exhibited anticancer activity by targeting the E3 ligase receptor DCAF15, with RBM39 as the only known substrate. Exploration of additional substrates of E7070 would facilitate elucidation of its mechanism of actions. To this end, we used a strategy combing pSILAC method with two complementary digestion approaches (LysC-Trypsin and LysN-LysArgiNase) to accurately monitor the protein turnover and increase the depth of proteome profiling. Systematically, we showed that E7070 treatment changed turnover rates of 868 proteins (1.5 fold change and p-value < .05). Several proteins displayed accelerated turnover indicating they were potential new substrates of E7070, among which, pre-mRNA splicing factor 39 (PRPF39) had been reported to be overexpressed in certain cancers. We further demonstrated that PRPF39 was ubiquitinated and degraded by E7070 in a DCAF15-dependent manner, and represented a new bona fide substrate of E7070. The degradation of PRPF39 might also be contributed to the anticancer activity of E7070. Significance: Identification of degraded substrates is difficult because protein abundance is a comprehensive result regulated by protein production and degradation at the same time. Pulsed SILAC (pSILAC), a method to measure protein turnover, would provide higher sensitivity than total protein quantification. In addition, some peptide sequences are not amenable to MS analysis after LysC-Trypsin digestion. LysN-LysargiNase, as a mirror protease combination of LysC-Trypsin, can be complementary for peptide identification with LysC-Trypsin. By combining pSILAC with two complementary digestion approaches (LysC-Trypsin and LysN-LysArgiNase), we systematically investigated E7070-dependent protein degradation. As a result, we found several potential degradation substrates of E7070 including PRPF39. Further, by exploiting a series of biological assays, we demonstrated that E7070 can lead to the ubiquitination and proteasomal degradation of PRPF39 by promoting the recruitment of PRPF39 to the CUL4-DCAF15 E3 ubiquitin ligase.
资助项目	Special Project on Precision Medicine under the National Key RD Program[2017YFC0906600] ; National Natural Science Foundation of China[81872888] ; National Natural Science Foundation of China[81773018] ; National Science & Technology Major Project Key New Drug Creation and Manufacturing Program[2018ZX09711002-004]
WOS关键词	SULFONAMIDE ANTICANCER AGENT ; GENE ONTOLOGY ; PHASE-II ; E7070 ; IDENTIFICATION ; DEGRADATION ; TRYPSIN
WOS研究方向	Biochemistry & Molecular Biology
语种	英语
出版者	ELSEVIER
WOS记录号	WOS:000498278700013
内容类型	期刊论文
源URL	[http://119.78.100.183/handle/2S10ELR8/282026]
专题	中国科学院上海药物研究所
通讯作者	Liu, Bin; Tan, Minjia
作者单位	1.Hubei Polytech Univ, Hubei Key Lab Kidney Dis Pathogenesis & Intervent, Sch Med, Huangshi 435003, Hubei, Peoples R China 2.Shanghai Univ, Sch Life Sci, Lab Noncoding RNA & Canc, Shanghai 200444, Peoples R China 3.Chinese Acad Sci, Chem Prote Ctr, Shanghai Inst Mat Med, State Key Lab Drug Res, Shanghai 201203, Peoples R China 4.Univ Chinese Acad Sci, Beijing, Peoples R China
推荐引用方式 GB/T 7714	Jia, Xinglong,Pan, Lulu,Zhu, Mingrui,et al. pSILAC method coupled with two complementary digestion approaches reveals PRPF39 as a new E7070-dependent DCAF15 substrate[J]. JOURNAL OF PROTEOMICS,2020,210:11.
APA	Jia, Xinglong.,Pan, Lulu.,Zhu, Mingrui.,Hu, Hao.,Zhai, Linhui.,...&Tan, Minjia.(2020).pSILAC method coupled with two complementary digestion approaches reveals PRPF39 as a new E7070-dependent DCAF15 substrate.JOURNAL OF PROTEOMICS,210,11.
MLA	Jia, Xinglong,et al."pSILAC method coupled with two complementary digestion approaches reveals PRPF39 as a new E7070-dependent DCAF15 substrate".JOURNAL OF PROTEOMICS 210(2020):11.