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

Highlights

• pSILAC with two complementary digestion systems can monitor the protein turnover and increase the depth of proteome profiling

• Several potential substrates of E7070 were identified including RBM39 and PRPF39

• PRPF39 was ubiquitinated and degraded by E7070 in a DCAF15-dependent manner

• The degradation of PRPF39 might also be contributed to the anticancer activity of E7070

Abstract

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.

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 CUL4^DCAF15 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.