Thiostrepton Interacts Covalently with Rpt Subunits of the 19S Proteasome and Proteasome Substrates

Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

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Citation Sandu, Cristinel, Nagaranjan Chandramouli, Joseph Fraser Glickman, Henrik Molina, Chueh-Ling Kuo, Nikolay Kukushkin, Alfred L Goldberg, and Hermann Steller. 2015. “Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates.” Journal of Cellular and Molecular Medicine 19 (9): 2181-2192. doi:10.1111/jcmm.12602. http:// dx.doi.org/10.1111/jcmm.12602.

Published Version doi:10.1111/jcmm.12602

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:22856985

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA J. Cell. Mol. Med. Vol 19, No 9, 2015 pp. 2181-2192

Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

Cristinel Sandu a, Nagaranjan Chandramouli b, Joseph Fraser Glickman c, Henrik Molina b, Chueh-Ling Kuo d, Nikolay Kukushkin d, Alfred L. Goldberg d, Hermann Steller a, *

a Howard Hughes Medical Institute, Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY, USA b Proteomics Resource Center, The Rockefeller University, New York, NY, USA c High Throughput Screening Resource Center, The Rockefeller University, New York, NY, USA d Department of Cell Biology, Harvard Medical School, Boston, MA, USA

Received: November 20, 2014; Accepted: April 3, 2015

Abstract

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell- based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IjBa degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.

Keywords: thiol protein degradation protein chemical modiﬁcation proteasome ubiquitin

Introduction

We identified Thiostrepton (Thsp) as a strong hit in a cell-based of the 70S ribosome, in a cleft between L11 subunit and H43/H44 of screen for modulators of Drosophila Inhibitor of Apoptosis Protein 1 the 23S rRNA, and in this way obstructs the recruitment and turnover (DIAP1) stability in human cells. Inhibitor of apoptosis proteins of the elongation factor EF-G [8–10]. In mammals, Thsp does not (IAPs), including DIAP1 are E3 ubiquitin ligases that regulate the lev- block the cytoplasmic protein translation, because of the sequence els of important proapoptotic factors such as caspases, by binding difference in 28/23S rRNA that prevents Thsp binding [11]. Reminis- and targeting them for degradation by the ubiquitin -proteasome cent of its function in bacteria, Thsp was shown, however, to inhibit pathway (UPP) [1–3]. mammalian mitochondrial translation [12]. Consistent with these Thiostrepton is a natural antibiotic produced by microorganisms observations it was shown that Thsp reduces the levels of mitochon- of the Streptomyces genus [4–6]. It is a large molecule (1.66 kD), drial cytochrome oxidase I, triggers reactive oxigen species (ROS) (in translated by the ribosome and following leader sequence cleavage it combination with arsenic trioxide) where this effect can be rescued undergoes extensive post-translational modifications [7]. In bacteria, by free radical scavenger N-acetyl-L-cysteine (NAC) [13]. Thsp blocks protein translation through binding to the GTPase centre Using gene expression profiling, it was shown that Thsp induces oxidative and proteotoxic stress, as revealed by the transcriptional up-regulation of heat shock, oxidative stress and endoplasmic reticu- *Correspondence to: Dr. Hermann STELLER lum (ER) stress genes [14]. Oxidative and proteotoxic stress effects E-mail: [email protected] were relieved by treatment with the anti-oxidant NAC. Using a mass

doi: 10.1111/jcmm.12602 ª 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. spectrometry approach, we also showed that proteasome inhibitor lished from a single cell that was resistant to Puromycin treatment, MG-132 and Thsp induce up-regulation of heat shock proteins, con- following an established procedure [30]. Dual-colour DIAP1 sensor cells sistent with a proteotoxic stress response [15]. Recently, it was sug- were generated by stable cotransfection of HEK293 with pcDNA3.1(+) D – gested that the mechanism of oxidative stress induced by Thsp is Puro-DIAP1 R-mCherry (DIAP1 corresponding to residues 1 320 fused caused by the inactivation of mitochondrial peroxiredoxin-3, thus dis- to mCherry gene) and Rpr-HA construct cloned in pIRES2-EGFP vector (Invitrogen, Carlsbad, CA, USA). Thiostrepton EC was determined in abling an important anti-oxidative network [16]. Thiostrepton trig- 50 using this dual -colour DIAP1 sensor cell line. Increasing amounts of gered a protein mobility shift change in peroxiredoxin-3, and it was Thsp (0–20 lM) was incubated with 3000 cells in a 40 ll culture vol- proposed that this is caused by a covalent adduct formation, similar ume (384 well plates) for 18 hrs. The plates were scanned using Imag- to the interaction between Thsp and TipAS protein from Streptomyces eXpress Velos Laser Scanning Cytometer (Molecular Devices, lividans [17]. Thiostrepton was also shown to inhibit FoxM1 function Sunnyvale, CA, USA) to collect 5-lm resolution red and green fluores- [18–21]. cence images. The images were segmented using the ImageXpress Ve- It has been reported that Thsp acts as an inhibitor of the 20S los analysis software (Molecular Devices) to recognize individual proteasome chymotrypsin activity [22, 23]. However, the mechanism fluorescent particles on both channels. The data for each concentration * by which Thsp inhibits proteasome activity remains unknown. The were represented as total fluorescence (TF) red/TF green 100. For ability of Thsp to inhibit the proteasome is of potential clinical rele- screening purposes this fluorescence number above was normalized against the fluorescence number of dimethyl sulfoxide (DMSO) (0%) vance as the proteasome is the major cellular protein degradation fac- and that of 10 lM MG-132 (100%). The proteasome sensor consists of tory and proteasome inhibitors are currently used for human therapy HEK293 cells transfected with pZsProSensor-1 plasmid (Clontech, Palo (Bortezomib/Velcade) or are in clinical trials [24, 25]. Bortezomib is Alto, CA, USA). Positive colonies were selected based on detectible used for the treatment of relapsed multiple myeloma and mantle cell green fluorescence. lymphoma. Identification of novel proteasome inhibitors may provide alternatives to current therapy that are potentially less toxic or target the proteasome by a distinct mechanism. Proteins, compounds, antibodies Thiostrepton induces apoptotic cell death in various human cancers, including breast, melanoma, leukaemia, liver cancer or Rpr protein (residues 1–65) followed by GSSHHHHHH tag was malignant mesothelioma [14, 16, 19, 20, 26, 27]. Cancer cells appear purified as described previously [29]. RprPep (AVAFYIPDYPYDVVPD- to be more sensitive to Thsp than normal cells, as it was shown for YATSCHPKTGRKSGKYRKPSQ), at 95% purity was synthesized by (ELIM melanoma cells versus primary melanocytes [14, 15]. Moreover, Bio, Hayward, CA, USA). All the compounds in this work, otherwise speci- although Thsp induces proteotoxic stress in both melanoma and pri- fied, were dissolved in DMSO. Compounds were purchased from com- mary melanocytes, only cancer cells undergo cell death [15]. Thio- mercial inventories as follows: MG-132 (Calbiochem, San Diego, CA, USA), Thsp (Tocris Cookson Inc. (Ellisville, MO, USA)). Comp-3 was strepton was not considered for human therapy because of its poor kindly provided by Dr. Patrick G. Harran, UCLA or synthesized by Ouathek solubility and unfavourable pharmacodynamics. However, because of Ouerfelli and Barney Yoo at the Organic Synthesis Core Facility of the its significant anti-cancer properties and with increasing clarification MSKCC. The antibodies used in this work were purchased as follows: rab- of its mechanisms of action, Thsp remains an interesting molecule bit anti-DIAP1 (laboratory collection), chicken anti-Rpr (laboratory collec- that may have possible clinical utility. Currently, Thsp is used in mam- tion), rabbit anti-GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, mals as topical medication in veterinary medicine for the treatment of USA), rabbit anti-Ups24 (Protein Group Inc., Chicago, IL, USA), anti-HA- mastitis and dermatological disorders [28]. HRP (Roche, Indianapolis, IN, USA), mouse anti-polyubiquitin, FK2 clone In this study, we found that Thsp acts as an inhibitor of the 19S (Millipore, Billerica, MA, USA), mouse anti-p53 (Santa Cruz Biotechnol- proteasome. Thiostrepton forms adducts with human proteins and its ogy, Inc), rabbit anti-IjBa (Santa Cruz Biotechnology, Inc), mouse anti- ability to interact covalently with cysteine residues is essential for p21 (Cell Signalling Technology, Danvers, MA, USA), rabbit anti-Mcl1 (Cell Signalling), mouse anti-GAPDH (Abcam, Cambridge, MA, USA), anti- proteasome inhibition. We characterized the nature of the adducts Rpt1 to anti-Rpt6 (Enzo Life Sciences, Farmingdale, NY, USA). Purified and show that Thsp bridges between Rpt proteasome subunits and bovine 26S proteasome (UBP Bio, Aurora, Co, USA), purified rabbit 20S proteasome substrates. These findings suggest a novel mode of pro- proteasome (Boston Biochem, Cambridge, MA, USA), human and bovine teasome inhibition, which occurs at the substrate unfolding/transloca- 19S regulatory particle (UBP Bio). tion step.

Western blot and immunoprecipitation of DIAP1 Materials and methods complexes

Cell culture, transfections and plasmids Cells were lysed in lysis buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 0.5% NP-40) and extracts were cleared by centrifugation at 20,817 9 g. 15 lg cell extract were regularly used for detection of proteins by WB. DIAP1 sensor cell line Immunoprecipitation of DIAP1DR-YFP complexes from extracts derived HEK293 cells were stably cotransfected with pcDNA3.1(+)Puro- from DIAP1 sensor cells were achieved using a GFP puriﬁcation DIAP1DR-YFP (DIAP1 corresponding to residues 1–320 fused to YFP) kit (Miltenyi Biotech, San Diego, CA, USA), following manufacturer construct and pcDNA3.1(+)Puro-Rpr-HA [29]. Sensor cells were estab- instructions.

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Proteasome activity determination Mass spectrometry analysis of Rpr complexes

Proteasome activities were determined using purified bovine 20S pro- Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-ToF) teasome (Boston Biochem) or purified bovine 26S proteasome (UBP mass spectrometry analysis (Voyager-DE-STR; Applied Biosystems, Bio) and using synthetic proteasome substrates. The three specific Carlsbad, CA, USA) was performed in linear mode using a standard activities (chymotrypsin-, trypsin- and caspase-like) of the proteasome Nitrogen laser. Recrystallized alpha-cyano-4-hydroxycinnamic [31] were determined using either fluorogenic tri-peptide substrates (Sigma-Aldrich, Steinheim, Germany) was used as MALDI matrix. Sam- (Bachem, Torrance, CA, USA) or a chemiluminiscent Protesome-GloTM ples of Rpr (0.3 lg/ll) alone or reacted with Thsp, respectively, were chemotrypsin-like assay (Promega, Madison, WI, USA). A typical reac- each diluted 1:1 with formic acid (‘High Purity Grade’, Sigma-Aldrich). tion consisted of 1.3 nM purified proteasome, 20 lM substrate in a vol- One microlitre of each sample was spotted on to a standard stainless ume of 40 ll. The compounds were added to the reaction at a 5 or steel MALDI target followed by the addition of 2 ll matrix solution. The 10 lM final concentration. The reaction is performed in a white 96 well spotted solution was gently mixed with the tip of a pipette for better plate with clear bottom. The hydrolysis of fluorescent peptides was mixing and crystallization. Mass spectra were acquired using an acceler- monitored continuously for 30 min. and the rates of hydrolysis were ation voltage of 20 kV and an extraction delay of 200–1200 nsec. For shown in the figures. The chemi-luminescence reactions were measured each spot, spectra from 500 laser shots were summed. All measure- using a Spectramax M2 plate reader (Molecular Devices). A time-depen- ments were performed in replicate. dent kinetic was performed for 30 min. (10 reads every other 3 min.) and data were analysed using Softmax Pro 4.7.1. (Molecular Devices). Proteasome activity is represented as Vmax, calculated from the slope Molecular structure visualization and distance (RTL/sec.) of the reaction. measurements

In vitro assay for Rpr-Thsp adduct formation Distance measurements, structural images rendering and snap-shots acquisition of Thsp (PDB: 2L2W), yeast 26S proteasome (PDB: 4B4T) or archaeal proteasome (PDB: 3IPM) were achieved using molecular l The assay consisted of 10 l reaction containing either 350 pmol Rpr visualization software Pymol Software (DeLano Scientiﬁc LLC, Palo Alto, and 2.5 nmol Thsp or 350 pmol Rpr and 2.5 nmol Thsp and 7.5 nmol CA, USA). L-cysteine. The reaction buffer was 25 mM Tris, pH 8.0, 50 mM NaCl. The reaction was incubated at 37°C for 0–3 hrs. After the determined time, the reactions were analysed by SDS-PAGE, WB or by mass spectrometry. Formation of RprPep-Thsp adduct was done similarly in a Results 10 ll reaction consisting of 2.38 nmol RprPep and 5 nmol Thsp in 25 mM Tris, pH 8.0, 50 mM NaCl. The reaction was incubated for 3 hrs at RT and the formation of RprPep-Thsp was validated by Thiostrepton stabilizes a labile DIAP1-based SDS-PAGE. protein sensor in human cells

In vitro We identified Thsp in a high-content cell-based small molecule screen ubiquitination of RprPep and RprPep- for modulators of DIAP1 stability in human cells. The IAP-antagonist Thsp adducts Rpr was previously shown to bind DIAP1 and stimulate its self-ubiquitination and degradation by the UPP [29, 32, 33]. Furthermore, Rpr 1.1 nmol of RprPep or RprPep-Thsp adduct (prepared as above), was can stimulate degradation of DIAP1 lacking the E3 ligase activity incubated with 1.0 lg E1, 1.0 lg E2, 1.0 lg Flag-DIAP1 and 3 lg (DIAP1DR) in human cells [29]. On the basis of these observations, Ubiquitin in 20 ll reaction. The reaction buffer was 25 mM Tris, pH we designed a fluorescent reporter cell line that is sensitive to protea- 7.5, 50 mM NaCl, 4 mM ATP and 5 mM MgCl. The reaction was incu- some inhibition. Specifically, we generated HEK293 cells stably trans- bated for 100 min. at RT, and flash frozen until further use. fected with both fluorescent DIAP1DR and Rpr-HA (Fig. 1A). Using this line, we performed a small-molecule screen for modulators of D In vitro degradation of RprPep by purified 26S DIAP1 R degradation. Chymotrypsin proteasome inhibitor MG-132 and a peptido-mimetic IAP-antagonist (Comp-3) caused a fluores- proteasome cence increase in this assay and were used as positive controls [34]. Thiostrepton showed a strong activity in the sensor cells and Degradation reactions consisted of 0, 0.4, 1.0 and 2.0 pmol bovine 26S increased DIAP1DR-YFP fluorescence very similar to MG-132 proteasome (UBP Bio) and 55 pmol ubiquitinated RprPep or RprPep- (Fig. 1B). Thiostrepton is a known bioactive natural compound that is Thsp adducts in a final volume of 10 ll. The reaction buffer was ribosomally produced by Gram-positive bacteria of the genus Strepto- 25 mM Tris, pH 7.5, 50 mM NaCl, 4 mM ATP and 5 mM MgCl, myces, and which undergo extensive post-translational modifications 250 lM DTT. Following incubation at RT for 40 min., the reactions were stopped by the addition of 5 ll sample buffer and subsequent boiling at [7]. Thiostrepton contains four reactive groups, three dehydroalanine 95°C. The samples were separated by SDS-PAGE, and RprPep and Rpr- (DHA) and one dehydrobutyrine (DHB), which can interact covalently Pep-Thsp adduct deubiquitination or degradation were monitored by with Cysteine residues, as it was demonstrated with Streptomyces WB using an ubiquitin antibody (FK2) or Rpr antibody. TipAS protein [17] (Fig. 1C). We have quantified Thsp activity and

ª 2015 The Authors. 2183 Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. found that it is a potent compound with an EC50 of 4.9 lM in DIAP1 bition (Fig. 2A). Like MG-132, Thsp triggers accumulation of sensor cells, slightly weaker than MG-132 with an EC50 of 1.1 lM polyubiquitinated species and labile endogenous p21 in MIA-PaCa 2 (Fig. 1D). To understand the mechanism of Thsp-induced DIAP1DR- cells (Fig. 2B), further supporting a role in proteasome inhibition and YFP fluorescence increase, we determined DIAP1DR ubiquitination is consistent with previous reports [14, 22]. Next, we tested the ability status in IP fractions from DIAP1 sensor cells treated with of Thsp to block the three catalytic activities in the context of purified compounds (Fig. 1E). Interestingly, Thsp induces accumulation of 20S proteasome, using small synthetic-labelled substrates (Fig. 2C). polyubiquitinated DIAP1DR, similar to MG-132 but distinct from Thiostrepton decreases the chymotrypsin activity (hydrophobic sub- Comp-3, an effect that is consistent with a role of Thsp in UPP strates) of the 20S proteasome, while the caspase activity (acidic sub- inhibition. strates) is not affected. Surprisingly, Thsp appears to increase the trypsin-like activity (basic substrates) of the 20S proteasome by four- fold. We also tested the effect of Thsp on the chymotrypsin activity in Thiostrepton blocks proteasome function in the context of purified 26S proteasome (Fig. 2D). Interestingly, Thsp human cells does not block the chymotrypsin activity in 26S proteasome, which is at odds with the observations involving 20S proteasome. As a control, To further understand the role of Thsp in UPP inhibition, we have chymotrypsin activity inhibitor MG-132 blocks activity in this assay tested whether Thsp can signal in a proteasome sensor cell line very efficiently. The overall chymotrypsin activity of 26S proteasome (Fig. 2A). The sensor is based on a fluorescent and unstable chimeric is much higher than that of 20S proteasome, which is explained by protein (ZsGreen-MODC) that is reportedly degraded by the protea- the difference in the state of 20S gate opening between these proteasome [35–37]. The sensor is functional and specific as it is respon- some species [38]. Thiostrepton was previously reported to inhibit sive to a proteasome inhibitor (MG-132) but not to IAP-antagonist the chymotrypsin activity of the proteasome [22, 23]. However, our Comp-3 (Fig. 2A). Thiostrepton stimulates accumulation of fluores- in vitro assays using purified 20S and 26S proteasomes revealed that cence in these cells, indicating that it plays a role in proteasome inhi- the effects are more complex.

A B

CD E

Fig. 1 Thiostrepton induces accumulation of a labile reporter in human cells. (A) Schematic representation of a fluorescence-based DIAP1-sensor, represented by HEK293 cells stably cotransfected with DIAP1DR-YFP fusion and Rpr-HA. IAP-antagonist Rpr binds to DIAP1 and triggers its ubiquitination and degradation. (B) Fluorescence micrographs of DIAP1-sensor cells, showing DIAP1DR-YFP fluorescence response following DMSO, MG-132, Comp-3 (synthetic IAP-antagonist) and Thsp treatment for 18 hrs. (C) Chemical structure of Thsp with highlighted reactive dehydroalanine residues (DHA3/DHA16/DHA17) and the dehydrobutyrine residue (DHB8). (D) Diagram showing the determination of EC50 for MG-132 and Thsp in DIAP1-sensor cells. (E) Thsp triggers accumulation of polyubiquitinated DIAP1 species. DIAP1DR-YFP was immunoprecipitated from IAP-sensor cells treated with the specified compounds, and the ubiquitination status and DIAP1 levels were determined by WB with anti-ubiquitin and anti- DIAP1 antibodies.

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BC D

Fig. 2 Effect of Thiostrepton on proteasome activity in human cells and in vitro.(A) Left, schematic representation of the ZsGreen-MODC sensor cell line. Human HEK293 cells were stably transfected with a construct that entails Zoanthus sp. GFP gene fused to mouse ornithine decarboxylase deg- ron (MODC). Right, microscopy images showing the fluorescence of the labile GFP-MODC reporter following DMSO, MG-132, Comp-3 and Thsp treatment for 21 hrs. (B) WB detection of polyubiquitin species and p21 accumulation in extracts of MIA-PaCa-2 cells treated with DMSO, MG-132 and Thsp for 20 hrs. (C) Effect of Thsp on catalytic activities (chymotrypsin-, trypsin- and caspase-like) of the purified bovine liver 20S proteasome. (D) Effect of Thsp and MG-132 on the activity of purified bovine 26S proteasome.

Thiostrepton can bind covalently to proteins in and oligomers was also observed for DIAP1 (Fig. 3C) and Rpr human cells (Fig. 3D), which are expressed ectopically in HEK293 as part of the DIAP1 sensor. Given the presence of reactive DHA and DHB residues in Thsp and Since Rpr appears to be modified by Thsp in cells, we decided to its reported ability to interact with proteins such as TipAS, we investigate this interaction in vitro, as a model of Thsp–protein inter- investigated whether Thsp inhibits protein degradation by interact- action. Rpr has only 65 amino acids, which makes it easier to ana- ing covalently with cellular proteins [17]. Being a large molecule, lyse by mass-spectrometry (MS) and contains a single Cysteine the interaction with a protein might cause a shift in protein size that residue (Cys 49) that simplifies the possibilities of potential interac- can be detected by WB. To test this hypothesis, we incubated tions with Thsp. As such, purified Rpr was examined by Coomassie extracts of HEK293 cells with either 75 lM or 7.5 lM Thsp for staining, which confirmed its size and also the presence of protein 1 hr or just briefly for 3 min., and tested for changes in these dimers in small amounts (Fig. 3E). Then, purified Rpr was analysed extracts by WB using available antibodies. We noticed that certain by MALDI-ToF MS, which confirmed the expected MW and the pres- proteins such as p53 or Usp24 either show the formation of addi- ence of Rpr dimers (Fig. 3F). In parallel, purified Rpr was incubated tional higher molecular bands or have undergone complete migra- with Thsp in a time-dependent fashion and the samples were analy- tion shifts (Fig. 3A). This reaction appears to develop in time, as sed as above. Thiostrepton induced formation of novel bands besides short incubation does not lead to detectable migration shifts Rpr, which are consistent with potential Rpr-Thsp and (Rpr)2-Thsp (Fig. 3A). To determine whether these effects occur in living cells, adducts by Coomassie staining (Fig. 3G). Indeed, the formation of we treated HEK293 with Thsp in a time-dependent fashion and Rpr-Thsp and (Rpr)2-Thsp adducts species was confirmed by tested by WB the appearance of p53 higher molecular bands, as an MALDI-ToF MS (Fig. 3H). The existence of protein-Thsp adducts at indicator of Thsp (re)activity. This is indeed the case, as the forma- 1:1 ratio has been previously demonstrated with Streptomyces pro- tion of p53 higher molecular bands became distinct at 4–8 hrs tein TipAS, yet this is the first demonstration of a Protein–Thsp–Pro- treatment (Fig. 3B). Appearance of bands consistent with dimers tein adduct (2Protein: 1Thsp ratio), which explains the appearance of

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Fig. 3 Thiostrepton interacts covalently with proteins in human cells. (A) WB detection of Usp24 and p53 in HEK293 cell extracts incubated with different concentrations of Thsp, for different times. Note the formation of p53 and Usp24 dimers/multimers, an effect that is dependent on Thsp concentration and incubation time. (B) Time-dependent formation of p53 multimers in HEK293 cells treated with Thsp (10 lM), as detected by WB. (C) WB detection of DIAP1DR-YFP in DIAP1-sensor cells treated with DMSO, MG-132, IAP-antagonist (Comp-3) and Thsp for 21 hrs. Besides DIAP1DR-YFP accumulation, Thsp also triggers formation of DIAP1 dimers and multimers. (D) WB detection of Rpr-HA and p21 in DIAP1-sensor cells treated with DMSO, MG-132 and Thsp for 21 hrs. Thiostrepton -dependent formation of Rpr dimers/multimers becomes apparent. (E) Left, Coomassie-stained SDS-PAGE gel showing purified Rpr protein incubated at RT for different times. Right, cartoon of monomeric Rpr with known structural elements, as well as cartoon of Rpr dimer. (F) MALDI–ToF analysis of purified Rpr protein, which confirms the MW of Rpr and Rpr dimer. (G) Left, Coomassie-stained SDS-PAGE gel showing the apparent formation of Rpr–Thsp adducts following incubation of Rpr with Thsp, in a time- dependent fashion. Right, schematic model of Rpr–Thsp adduct and of Thsp bridging between two Rpr molecules, (Rpr)2-Thsp. (H) MALDI–ToF confirmation of Rpr–Thsp and (Rpr)2–Thsp adducts formation. protein dimers observed in human cells following Thsp treatment dicted. Furthermore, we found that the ability of Thsp to block pro-

[17]. Despite its abundance in SDS-PAGE, detection of the (Rpr)2- teasome function in human cells (as visualized by fluorescence Thsp adduct by MALDI–ToF MS has been difficult, and only solubili- accumulation in DIAP1 and proteasome sensor cells) is quenched zation of the sample in 50% formic acid has enabled its unequivocal by the addition of L-Cysteine (Fig. 4B). This suggests that Thsp detection (Fig. 3H). needs to bind covalently to Cysteine residues to block the proteasome function. Subsequently, we asked whether Thsp is a rapid or slow inhibitor of proteasome function in the cell. To this end, we Covalent binding of Thiostrepton to proteins followed the proteasome-mediated IjBa degradation in HEK293 blocks proteasome function interleukin (IL)-1R cells, pre-treated with MG-132 or Thsp and then activated with IL-1 (Fig. 4C). Interleukin-1 treatment leads to IjBa Thiostrepton can interact covalently with Rpr. Next, we showed that degradation as part of signalling leading to NF-jB activation [39]. the formation of Rpr-Thsp adducts in vitro is counteracted by the Thirty minute preincubation of cells with MG-132 is sufficient to pre- supplementation of the reaction with excess L-Cysteine (Fig. 4A). vent IjBa degradation (Fig. 4C). By comparison, Thsp achieves the This is an indication that Thsp interacts with Cys49 in Rpr, as pre- same effect only after 4 hrs of pre-incubation and at a concentration

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10-fold higher than MG-132 (Fig. 4C). Interestingly, the slow rate of status in cell extracts and the levels of labile Mcl-1 protein. MG-132 proteasome function impairment by Thsp, correlates with the treatment for 4 hrs induced the accumulation of polyubiquitinated appearance of p53-Thsp adducts (Fig. 4C, lower panel) and is in species and Mcl-1 as expected. However, these effects were agreement with previous observations of Thsp–protein adducts reversed almost completely 4 hrs after MG-132 withdrawal, which which are formed in a time-dependent fashion (Fig. 3A). Covalent indicates that the proteasome function was restored in these cells attachment to proteins and slow proteasome inhibition, suggest the (Fig. 4D). When Raji cells were incubated with Thsp for 4 hrs, we possibility that Thsp is an irreversible inhibitor of proteasome func- observed the accumulation of polyubiquitinated species and a slight tion. To address this question, we treated Raji cells with CHX (to increase in Mcl-1. However, 4 hrs after Thsp withdrawal, the prevent de novo protein production), CHX/MG-132 or CHX/Thsp polyubiquitination remained strong and Mcl-1 levels continued to combinations. Four hours after treatment, the drugs were washed increase, which is in contrast to MG-132 (Fig. 4D). Collectively, off the cells and the cells were further incubated for 1–6 hrs. As these observations are consistent with an irreversible action of Thsp markers for proteasome function we monitored the ubiquitination on proteasome function in the cell.

A B

Fig. 4 Proteasome inhibition is dependent on Thiostrepton ability to bind covalently to proteins. (A) Time-dependent Rpr–Thsp adducts formation is prevented by L-Cysteine supplementation. Rpr was incubated in vitro with Thsp or Thsp/L-Cysteine combination and the formation of Rpr adducts was monitored by WB with Rpr-specific antibody. This is consistent with Thsp binding covalently to the Cysteine residue of Rpr (Cys49), a process that is blocked by addition of excess L-Cysteine. (B) Microscopic images showing the fluorescence response of DIAP1 sensor cells and proteasome sensors cells following treatment with Thsp or combination Thsp/L-Cysteine. Excess L-Cysteine blocks Thsp-induced accumulation in sensors’ fluorescence (or blocks Thsp-induced proteasome inhibition), which is consistent with a model where Thsp covalent attachment to Cysteine residues is determinant for its proteasome inhibitory activity. (C) Thsp is a slow inhibitor of the proteasome activity in the cell when compared to chymotrypsin activity inhibitor MG-132, and the proteasome inhibition by Thsp correlates with formation of Thsp–p53 adducts in the cell. HEK293 IL-1R cells were treated with MG-132 or Thsp followed by stimulation with IL-1 (10 ng/ml) for 15 min., to trigger proteasomal degradation of IjBa (NF-jB activation). Persistence of IjBa in samples pre-treated with MG-132 or Thsp and subsequently with IL-1, is an indication of proteasome inhibition. IjBa stabilization in Thsp treated samples correlates with accumulation of p53 multimeric bands (an indicator of Thsp–protein adducts formation). (D) WB detection of polyubiquitination and Mcl1 accumulation in extracts of Raji cells. The cells were treated with CHX, CHX/MG-132 and CHX/Thsp for 4 hrs, washed and supplemented with fresh media and then harvested at different time-points (1–6 hrs). GAPDH was used as a loading control. The extent of polyubiquitination and Mcl-1 levels, 4 hrs after washing off MG-132 are largely reversed to non-treated state. For comparison, these effects remained steady or increased 4 hrs after Thsp wash off, which is consistent with a covalent-binding proteasome inhibition model.

ª 2015 The Authors. 2187 Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Thiostrepton attached to substrates precludes Thsp adducts are not degraded by 26S proteasome under the same substrate degradation by the proteasome conditions (Fig. 5D), suggesting that the Thsp may hinder sterically the substrate/peptide translocation through the channels/gates of the To understand how Thsp-modified substrates are processed by the proteasome. proteasome, we needed a substrate that can be modified by Thsp and that can be polyubiquitinated in vitro. Thus, we generated RprPep (Fig. S1) that is based on Rpr protein, in which the folded central heli- Thiostrepton interacts covalently with subunits of cal region of Rpr was replaced with an HA tag, to generate an essen- the 19S Base and proteasome substrates tially unfolded peptide. In vitro incubation with Thsp, led to efficient formation of RprPep-Thsp adducts (Fig. 5A). RprPep being smaller A biotinylated Thsp derivative was used previously to pull down than Rpr and unfolded, allowed the formation of additional oligomeric binding partners in extracts of human cells [21]. Among the many adducts (trimers, tetramers). We have previously established an in vi- potential candidates identified by MS there were several protea- tro assay for Rpr ubiquitination that could be used for RprPep ubiqui- some subunits. A closer inspection indicated that many of those tination as well [29]. As such, we have ubiquitinated in vitro RprPep proteins were Rpt subunits of the Base in the 19S proteasome and RprPep-Thsp adducts samples, and incubated them in vitro with regulatory particle (Fig. 6A). To address the potential Thsp interac- purified 26S proteasomes to determine how these samples are de- tion with Rpt proteasome subunits, we incubated purified bovine ubiquitinated. Interestingly, incubation with increasing amounts of 26S proteasome with Thsp, RprPep or its combination and tested 26S proteasome, showed a very similar deubiquitination pattern it by WB to verify whether any subunits of the Base (Rpt proteins) (Fig. 5B). This indicates that Thsp-modified substrates do not impair were modified. Remarkably, from the Rpt subunits tested Rpt1 the ability of the proteasome to deubiquitinate substrates. Next, we showed a clear protein shift that was formed only in the presence wanted to determine whether RprPep and the RprPep-Thsp adducts of Thsp and RprPep, which can only be explained by the forma- are degraded by the 26S proteasome. Incubation of RprPep with tion of a Rpt1–Thsp–RprPep adduct (Fig. 6B). To confirm this dis- increasing amounts of 26S proteasome led to peptide degradation in covery, we next tested the ability of Thsp to form adducts with a proteasome concentration manner (Fig. 5C). Interestingly, RprPep- the human 19S proteasome and the RprPep substrate. To this

Fig. 5 Proteasome substrate covalently modified by Thiostrepton is not degraded by 26S proteasome. (A) WB detection of a 37 amino acids chimeric Rpr peptide (RprPep) and RprPep-Thsp adducts formed in vitro, using a Rpr-specific antibody. (B) WB detection of purified 26S proteasome mediated deubiquitination of in vitro polyubiquitinated RprPep or Rpr- CD Pep-Thsp adducts. Thiostrepton covalently attached to a polyubiquitinated substrate does not interfere with the ability of 26S proteasome to deubiquitinate substrates. (C) WB detection of non-ubiquitinated RprPep degradation by purified 26S proteasome. (D) WB detection of non-ubiquitinated RprPep-Thsp adducts degradation by purified 26S proteasome. Thiostrepton modified RprPep is not degraded by 26S proteasome in vitro.

2188 ª 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J. Cell. Mol. Med. Vol 19, No 9, 2015

A BC

DEF

Fig. 6 Thiostrepton links protein substrates covalently to the 19S subunit of the proteasome. (A) Left, representation of the yeast 26S proteasome structure, based on reported PBD code: 4B4T. The 19S subunit consists of the Lid (top, green semi-transparent) and the Base (middle, multicolour). The 20S subunit (on the bottom, yellow semi-transparent) entails the proteolytic sites and represents a platform for 19S attachment. Right, top view representation of the 19S Base, with each of the 6 Rpt subunits distinctly coloured and labelled. (B) WB detection of Rpt1, Rpt3, Rpt4 and Rpt5 following incubation of bovine 26S proteasome with RprPep, Thsp or both. The data show the appearance of a new band above Rpt1, in samples containing 26S, RprPep and Thsp, which indicates that RprPep is linked covalently to the Rpt1 subunit via Thsp. (C) WB detection of Rpt1, Rpt2, Rpt3, Rpt4, Rpt5 and Rpt6 in assays containing human 19S regulatory particle incubated with RprPep, Thsp or both. Appearance of new bands indicates that Thsp attaches RprPep covalently to Rpt1 primarily, and weakly to other base subunits such as Rpt2 and Rpt4. (D) WB detection of Thsp adduct formation with RprPep and Rpt1 in the context of bovine 19S proteasome. Addition of excess L-cysteine blocks formation of RprPep–Thsp and that of a distinctive band above Rpt1 (red arrow), which is consistent with an Rpt1–Thsp–RprPep adduct. (E) Cartoon model of substrate degradation by 26S proteasome, which include deubiquitination mediated by the 19S Lid, substrate unfolding and translocation (mediated by the 19S Base) and subsequent substrate degradation (mediated by 20S). (F) Model of Thsp-induced proteasome inhibition, with Thsp binding to Rpt1 subunit and blocking the process of substrate unfolding and translocation. During this process, proteasome substrates may get covalently attached to Rpt subunits as well. Thiostrepton is shown in blue and is highlighted by a semi-transparent circle. end, puriﬁed human 19S was incubated as above with Thsp, Rpr- investigated whether Rpt1–Thsp–RprPep adduct formation can be Pep or its combination and the formation of adducts with Rpt su- quenched by the supplementation of the reaction with excess L- bunits was tested by WB. Following the testing of all Rpt subunits cysteine. For this purpose, we incubated bovine 19S proteasome of the Base and consistently with the ﬁndings in panel B, Thsp with Thsp, RprPep or both in the absence or presence of excess attaches the proteasome substrate RprPep primarily to Rpt1 sub- L-cysteine (Fig. 6D). Consistent with observation in panels B and unit of the 19S regulatory particle. Additional Rpt proteins such as C we saw the appearance of a band above Rpt1 only in the Rpt2 and 4 serve as weaker binding partners (Fig. 6C). Next, we presence of Thsp and RprPep, suggesting the formation of

ª 2015 The Authors. 2189 Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. Rpt1–Thsp–RprPep adducts. Interestingly the addition of excess L- clarifiy the nature of dimeric species that are observed in cells, as cysteine prevented the formation of this band, which strongly cor- being protein dimers bridged by Thsp (Fig. 3H). Despite DHA and relates with the inability of Thsp to interact with cysteine residues DHB propensity to interact with cysteine residues, we find that in Rpt1 and RprPep. The quenching effects of cysteine are spe- Thsp does not interact with many cysteine-containing proteins (not cific, as it blocked the formation of RprPep–Thsp adducts in the shown) and is not an unspecific DUB inhibitor as it does not out- absence or presence of bovine 19S (Fig. 6D). These data are con- compete a DUB specific probe (HA-Ubiquitin-VME) from interacting sistent with a model where Thsp blocks the proteasome function covalently with the cysteine residue in the active site of human by interacting with the Rpt subunits of the 19S proteasome, and DUBs (Fig. S3) [42]. These results argue for a defined selectivity potentially by covalent attachment of proteasome substrates to the of Thsp in substrate addition. Base of the 19S proteasome, during the process of substrate The ability of cysteine supplementation to quench the formation unfolding and translocation (Fig. 6E and F). of Protein–Thsp adducts as well as to restore the proteasome function in cells is consistent with the notion that Thsp needs to attach covalently to proteins to inhibit proteasome function. Furthermore, Discussion the slow rate of proteasome inhibition by Thsp andthe persistence of proteasome inhibition markers after Thsp withdrawal, indicate We identified Thsp as a strong inhibitor of DIAP1 degradation in that Thsp is an irreversible inhibitor of proteasome function in a high-content high-throughput screen. Thiostrepton inhibits UPP- cells. The proteasome chymotrypsin-like activity inhibitor Velcade/ mediated protein degradation in a variety of assays, including a Bortezomib is currently used in the clinic for the treatment of multi- cell-based proteasome reporter, accumulation of polyubiquitinated ple myeloma and mantle cell lymphoma. However, the development proteins, stabilization of DIAP1, Rpr, Mcl-1 and p21. Previous of resistance that is often observed following therapy suggests that in vitro studies using purified 20S proteasomes and synthetic novel approaches of proteasome inhibition, including the 19S regu- small peptide-like substrates suggested that Thsp is a chymotryp- latory particle, might be needed [25]. sin inhibitor of the proteasome [14, 22, 23]. However, in contrast A Thsp probe was previously used to pull down protein binding to its effects on chymotrypsin activity, Thsp activated the trypsin- partners from a human cell extract and revealed several proteasomal like activity of 20S proteasome, while the caspase-like activity proteins among a long list of potential interacting partners [21]. A clo- remained largely unaffected. In addition, Thsp did not block the ser inspection indicates that these proteins are subunits of the Base chymotrypsin activity when using purified 26S proteasomes, of the 19S proteasome regulatory particle. The 19S Base consists of unlike the chymotrypsin inhibitor MG-132. Therefore, we investi- 6 Rpt proteins (1–6) organized as heterodimers, which form a cylin- gated the mechanism by which Thsp blocks the proteasome activ- der with a central pore and anchors on top of the 20S particle [43, ity in more detail. We suggested that the ability of Thsp to 44]. The Rpt proteins are ATP-ases and function in substrate unfold- prevent protein degradation in vivo may be related to both its ing – translocation step – which occurs after substrate deubiquitina- large size (1.66 kD) and ability to bind covalently to cellular pro- tion. Interestingly, we discovered that Thsp is able to interact teins. An inspection of the tri-dimensional structure shows that covalently with proteasome Base subunits (primarily Rpt1) and the Thsp is 26 A long and 18.2 A wide [40] (Fig. S2A). The diameter proteasome substrate (RprPep), in the context of bovine 26S protea- of the 20S proteasome gate in open conformation is approxi- some and human 19S regulatory particle. These data provide direct mately 22.5 A towards the outside and 16.7 A towards the inside evidence for the interaction between Thsp and Rpt subunits of the [41] (Fig. S2B). Therefore, Thsp is slightly larger in size than the 19S proteasome, but also for the presence of proteasome–protea- 20S proteasome gate and presumably cannot enter the 20S pro- some substrates covalent adducts. Because only a fraction of Rpt1 teasome. One hypothesis is that Thsp may block protein break- was covalently linked to RprPep substrate, this cannot explain the down by preventing the entry of substrates that are covalently extent of proteasome inhibition by Thsp in the cells. However, these linked to this compound. To test this model, we used a 37 amino experiments cannot rule out that a larger fraction of Rpt1 is actually acid chimeric Rpr peptide (RprPep) that can be covalently linked modified by Thsp, but Thsp alone may not trigger a visible shift by to Thsp and ubiquitinated. Linkage of Thsp to this substrate did western blot (Rpt1 is a large protein, 50 kD). Taken together, our not interfere with deubiquitination, but it specifically rendered it findings are consistent with a model of irreversible 19S proteasome resistant to proteasomal degradation. This is consistent with the inhibition by Thsp. This is a novel mechanism of proteasome inhibi- idea that Thsp sterically prevents the substrates it is attached to tion by a natural product, which forms a covalent adducts with 19S from translocating through proteasome pores. proteasomal subunits and proteasome substrates, therefore blocking Thiostrepton contains reactive DHA and DHB residues and is the process of substrate unfolding and translocation. known to form adducts with the Streptomyces TipAS protein at 1:1 ratio (TipAS-Thsp) [17]. Here, we showed that proteins in human cells including p53, Usp24 or Drosophila proteins DIAP1 Acknowledgements and Rpr are modified by Thsp to trigger the formation of apparent dimers and other oligomeric species, as it was reported for Perox- This work was funded by internal funds from H. S.’s Laboratory. H. S. is an yredoxin-3 [16]. Importantly, this report is the first demonstration investigator of the Howard Hughes Medical Institute. This work was funded in of a protein–Thsp adduct at 2:1 ratio (Rpr2-Thsp). Our MS data part by NIH grant RO1GM60124 (to H.S.), and additional support for this

2190 ª 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. J. Cell. Mol. Med. Vol 19, No 9, 2015 project was provided by a grant from the Robertson Foundation. H.S. is an the chemical screen supervision. Mass spectrometry experiments investigator of the Howard Hughes Medical Institute. were done by N.C. and H.M. Experiments in Fig. 2C and other non-included experiments were performed by C-L.K and N.K. The Conﬂicts of interest study was supervised by A.L.G. and H.S.

The authors conﬁrm that there are no conﬂicts of interests. Supporting information

Author contribution Additional Supporting Information may be found in the online version of this article: C.S. designed the research study, performed the experiments, analysed the data, and C.S. and H.S. wrote the paper. J.F.G. did Data S1 Model for formation of RprPep-Thsp adducts.

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