pSILAC method coupled with two complementary digestion approaches reveals PRPF39 as a new E7070-dependent DCAF15 substrate
Graphical abstract
Introduction
Proteolysis based on the ubiquitin-proteasome system (UPS) is an important mechanism for post-translational homeostasis of the proteome. Ubiquitin (Ub) can be covalently attached to substrate proteins through a cascade involving three enzymes termed Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), and Ub-ligating enzyme (E3), with the E3 imparting substrate specificity [1]. The human genome encodes nearly 600 E3 ligases, including nine cullins (Cul1, 2, 3, 4A, 4B, 5, 7, PARC and APC2) with each utilizing a unique set of substrate-specific factors [2]. These ligases, collectively known as Cullin-RING ligases (CRLs), are the largest family of E3 ubiquitin ligases in eukaryotes [3].
The studies on the mechanism underlying the anticancer activity of thalidomide have provided a new strategy to target disease-driven proteins. Thalidomide can bind directly to cereblon (CRBN), a DDB1- and CUL4-associated factor (DCAF), to confer the E3 ubiquitin ligase complex to ubiquitinate and degrade key targets, such as the zinc finger transcription factors ZFP9, IKZF1, IKZF3 [4,5] and other targets lacking a zinc finger domain including SALL4 [6], CSNK1A1 [7] and GSPT1 [8]. Resembling thalidomide, E7070-treatement also represents a strategy to degrade key protein in cancer [9,10].
E7070 is an aryl sulfonamide drug which initially inhibits carbonic anhydrase [11]. Now it has been widely tested in patients with advanced-stage solid tumors because of its anticancer activity by downregulating cyclins A, B1 and CDK2 to impact cell cycle [12,13]. Patients treated with E7070 alone had no unacceptable toxicity, however, no >10% of patients presented a clinical efficacy [[14], [15], [16], [17]]. The mechanism of its selectivity is still not completely understood. Recently, a pre-mRNA splicing factor, RBM39 (also known as CAPERa), has been reported to be recruited to the CUL4DCAF15 E3 ubiquitin ligase by E7070 and degraded via proteasome in human cancer cell lines [9,10]. However, whether E7070 has additional DCAF15-dependent substrates is still unknown.
A common difficulty for the identification of degraded substrates is that protein abundance can also be regulated by transcriptional or translational response. Pulsed SILAC (pSILAC), an approach for measurement of protein turnover, would have higher sensitivity than total protein quantification [18,19]. In addition, consecutive use of LysC and trypsin is common in top-down proteomics [20,21]. However, several peptide sequences are not amenable to MS analysis after LysC-Trypsin digestion with cleaving mainly at the carboxyl side of lysine or arginine. LysN-LysargiNase, as mirror protease of LysC-Trypsin to cleave at the aminol side of the amino acids, can compensate peptide identification for LysC-Trypsin digestion [22].
Here, for accurately monitoring the protein turnover and increasing the depth of proteome profiling, we used pSILAC method together with the combination of LysC-Trypsin and LysN-LysArgiNase digestion approach to study E7070-dependent protein degradation. We found several potential new substrates of E7070 including another pre-mRNA splicing factor PRPF39. We have further demonstrated that E7070 induced the ubiquitination and degradation of PRPF39 in a DCAF15-dependent mechanism.
Section snippets
In vitro growth inhibition assay
Cytotoxic effect of E7070 (MedKoo Biosciences) was determined by the Cell Counting Kit-8 assay. Briefly, HCT116 (ATCC) cells were treated with E7070 at various concentrations and incubated for three days. The absorbance (optical density, OD) at 450 nm was measured to perform CCK8 analysis. The IC50 value was calculated by the concentration-response curve fitting using the four-parameter method.
Cell culture and protein extraction
HCT116 cells were cultured in “light” medium (DMEM medium with unlabeled L-arginine and l-lysine) at
Identification of new E7070 substrates through pSILAC method
To test the anticancer activity of E7070, the growth-inhibitory activity of E7070 against human colon cancer cell line HCT116 cells was performed, which showed a consistent anticancer activity (IC50 0.99 ± 0.37 μM) with a study published by the Han et al. [9] (Fig. 1A).
In order to identify the potential degraded substrates induced by E7070 systematically, we designed a two-time point pSILAC experiment (Fig. 1B). Cells were harvested at 12 h and 24 h after treatment with E7070 or DMSO and
Discussion
Despite the successful clinical application of drugs targeting protein degradation, a variety of adverse effects or low efficacy still exist, suggesting that there may be more unknown mechanisms. Therefore, discovery of unknown substrates can help us to better understand the mechanism of actions of drugs targeting CRL4 E3 ligase, such as thalidomide as well as E7070. Especially, for E7070, only one CRL4DCAF15 dependent substrate was reported, and thus we assumed that there could be more
Conclusion
In this study, we used pSILAC to accurately monitor the protein turnover and two complementary digestion approaches (LysC-Trypsin and LysN-LysArgiNase) to increase the depth of proteome profiling as well as quantification reliability. Altogether we found that E7070 treatment caused the accelerated turnover of several proteins including RBM39 and PRPF39. We further demonstrated that E7070 induced the ubiquitination and degradation of PRPF39 in a DCAF15-dependent mechanism by recruiting PRPF39 to
Author contributions
X.J, L.P and M.Z conducted most of the biochemical and cellular experiments with the help of H.H, L.Z, J.L and M.H under the supervision of B.L and M.T. X.J and L.P wrote the manuscript with the help of B.L and H.H, and M.T revised it. All authors read and approved the final manuscript.
Declaration of Competing Interest
The authors declared that they have no conflicts of interest.
Acknowledgements
This work was supported by the Special Project on Precision Medicine under the National Key R&D Program (No.2017YFC0906600), National Natural Science Foundation of China (81872888, 81773018), National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (No. 2018ZX09711002-004) to M.T.
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These people contributed equally.