ARTICLE Received 28 Jun 2016 | Accepted 23 Mar 2017 | Published 22 May 2017 DOI: 10.1038/ncomms15398 OPEN pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4CRBN ubiquitin ligase Jian An1,2, Charles M. Ponthier1, Ragna Sack3, Jan Seebacher3, Michael B. Stadler3,4, Katherine A. Donovan1,2 & Eric S. Fischer1,2 Thalidomide and its derivatives lenalidomide and pomalidomide (IMiDs) are effective treat- ments of haematologic malignancies. It was shown that IMiDs impart gain-of-function properties to the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) ubiquitin ligase that enable binding, ubiquitination and degradation of key therapeutic targets such as IKZF1, IKZF3 and CSNK1A1. While these substrates have been implicated as efficacy targets in multiple myeloma (MM) and 5q deletion associated myelodysplastic syndrome (del(5q)-MDS), other targets likely exist. Using a pulse-chase SILAC mass spectrometry-based proteomics approach, we demonstrate that lenalidomide induces the ubiquitination and degradation of ZFP91. We establish ZFP91 as a bona fide IMiD-dependent CRL4CRBN substrate and further show that ZFP91 harbours a zinc finger (ZnF) motif, related to the IKZF1/3 ZnF, critical for IMiD-dependent CRBN binding. These findings demonstrate that single time point pulse-chase SILAC mass spectrometry-based proteomics (pSILAC MS) is a sensitive approach for target identification of small molecules inducing selective protein degradation. 1 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA. 2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02215, USA. 3 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. 4 Swiss Institute of Bioinformatics, CH-4058 Basel, Switzerland. Correspondence and requests for materials should be addressed to E.S.F. (email: eric_fi[email protected]). NATURE COMMUNICATIONS | 8:15398 | DOI: 10.1038/ncomms15398 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15398 he ubiquitin proteasome system (UPS) constantly remodels simultaneously with applying the desired perturbation, such as the proteome to regulate a plethora of cellular processes1–3. treatment with a drug. Samples are subjected to shotgun TIn the UPS, ubiquitin is covalently coupled to a substrate proteomics utilizing liquid chromatography coupled tandem mass lysine by activity of an E1 (ubiquitin activating enzyme), E2 spectrometry (LC-MS/MS)29. The pre-existing (light amino acid (ubiquitin conjugating enzyme) and E3 (ubiquitin ligase) enzyme containing) and newly synthesized (heavy amino acid containing) cascade. Specificity in the UPS is largely conferred by E3 ligases, of protein species can be differentially quantified based on their which there are more than 600 in the human genome4. characteristic mass difference to derive heavy to light (H/L) protein Thalidomide and its derivatives lenalidomide and pomalidomide ratios30. In such an experimental design, and assuming constant (collectively known as IMiDs for immune-modulatory drugs), incorporation rate, logarithmic H/L ratios should increase linearly while initially marketed as sedatives found to cause severe over time. H/L protein ratios represent a quantitative measure of teratogenic side effects, are widely used to treat haematologic protein stability and differences between treated and control cells malignancies, including multiple myeloma (MM) and 5q can be used to identify drug-induced changes to protein turnover, deletion associated myelodysplastic syndrome (del(5q) MDS)5–8. such as induced degradation of target proteins. We demonstrate Lenalidomide binds to the ubiquitin ligase CRL4CRBN and exhibits that a single time point pSILAC experiment is sufficient to dual activity by inhibiting CRL4CRBN from ubiquitinating endo- identify CSNK1A1 along with new substrate candidates. We further genous substrates such as MEIS2 (refs 9,10), while simultaneously show that the zinc finger (ZnF) protein, ZFP91, a putative ubiquitin promoting the CRL4CRBN-dependent ubiquitination and ligase31,32,isabona fide lenalidomide-dependent CRL4CRBN degradation of IKZF1 (Ikaros), IKZF3 (Aiolos) and Casein kinase substrate. 1 alpha (CSNK1A1)10–14. IKZF1 and IKZF3 have been implicated in MM anti-proliferative effects of IMiDs, and CSNK1A1 has been shown to be the efficacy target of lenalidomide in del(5q)-MDS8. Results Recent reports have further demonstrated that lenalidomide Identification of novel CRL4CRBN targets through pSILAC MS. disrupts the ubiquitin-independent regulation of CD147 and We sought to explore a single time point pSILAC approach, MCT1 by CRBN15 and that glutamine synthetase (GS) is which would increase the depth of proteome profiling and ubiquitinated by CRL4CRBN in an IMiD-independent fashion16. significantly reduce the required machine time compared to multi However, the pleiotropic effects of IMiDs and the largely time point experiments. To understand the scopes and limitations unexplained adverse effects such as the profound teratogenicity of this approach, we performed a direct comparison with multi and neurotoxicity suggest that other substrates likely exist. To time point pSILAC and quantified the changes to total protein identify novel CRL4CRBN substrates regulated by lenalidomide, we abundance using tandem mass tags (TMT)33. performed proteomics studies in non-hematopoietic cell lines to To generate a reference data set for direct comparison, we avoid masking of results with the dominant presence of IKZF1/3 in first designed a multi time point pSILAC experiment. We hematopoietic tissues. used metabolic pulse labelling with heavy amino acids to Mass spectrometry-based methods have become standard for measure protein turnover in growing HEK293T cells treated the identification of E3 ubiquitin ligase substrates and were with 30 mM lenalidomide or dimethyl sulfoxide (DMSO) control successfully applied to various classes of ligases, such as the Cullin (Fig. 1a). The high concentration of lenalidomide was chosen RING ligase family SCF/CRL1 (refs 17–19). However, other to achieve maximum response in a short time window and ligase families have proven recalcitrant to commonly used thereby aid substrate identification. HEK293T cells were chosen methods such as affinity-purification mass spectrometry, di-Gly to bias the experiment towards targets beyond the well-studied proteomics or whole proteome relative quantification. Common transcription factors IKZF1 and IKZF3 that are found explicitly complications for the identification of ubiquitin ligase substrates in hematopoietic lineages34. Cells were collected in duplicates are secondary effects, such as transcriptional response or changes at three time points, 6 h (T6), 10 h (T10) and 16 h (T16) to translation. For profiling of protein stability-directed drugs, post treatment with lenalidomide or DMSO and parallel such as lenalidomide, most secondary effects can be reduced metabolic labelling. To prevent arginine to proline conversion, through relatively short drug treatments (o24 h) compared to a pervasive problem in SILAC, the cell culture medium was the typical treatment times for RNAi or genetic inactivation supplemented with excess unlabelled proline. Label swap (typically 448 h). However, faced with the challenge of relatively experiments at the T6 and T10 time points were performed small changes to total protein levels, we reasoned that an and analysed to exclude the possibility of SILAC label artefacts approach directed at measuring changes to protein turnover in (growth medium changing from H/L isotope label on treatment, response to drug treatment would provide increased sensitivity see also Supplementary Figs 1a–c and 2a–c). While we observe compared to relative quantification of total protein abundance. a small systematic offset in H/L protein ratios comparing forward Such a sensitive method would also be valuable to a growing field to reverse experiments (most pronounced at early time points), of drug development efforts directed at protein degradation20–23. the overall correlation between forward and reverse experiments Pulse-chase experiments, performed with 35S isotope labelling and random spot checks suggest that label artefacts can or through blockade of translation by treatment with cyclohex- be neglected for further analysis. Whole-cell lysates were imide (CHX), are considered the gold standard method pre-fractionated into 15 fractions by SDS–PAGE and tryptic to investigate protein stability. Modern mass spectrometry peptides subjected to LC-MS/MS analysis on an Orbitrap VELOS combined with stable isotope labelling (SILAC) can principally platform (see Methods section). Mass spectrometry data be used to perform pulse-chase experiments on a proteome-wide were analysed with the MaxQuant software35. We identified scale, and this has been applied to interrogate global protein 100,763 peptide sequences, which were assigned to 6,328 unique synthesis and decay rates24–28. However, such experiments protein groups (false discovery rate o1% on peptide and protein typically involve multiple time points. level; requiring min one unique peptide quantified for a protein Here we show that direct measurement of changes in protein to be included). stability in a single time point mass spectrometry experiment is a Polypeptides synthesized after drug treatment and metabolic sensitive and robust alternative to identify targets of lenalidomide. labelling will predominantly