Important Role of SUMOylation of Spliceosome Factors in Prostate

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Important Role of SUMOylation of Spliceosome Factors in Prostate Cancer Cells Donghua Wen, Zhijian Xu, Li Xia, Xinyi Liu, Yaoyao Tu, Hu Lei, Weiwei Wang, Tong-Dan Wang, Lili Song, Chunmin Ma, Hanzhang Xu, Weiliang Zhu, Guo-Qiang Chen, and Yingli Wu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr4012848 • Publication Date (Web): 15 Jul 2014 Downloaded from http://pubs.acs.org on July 17, 2014

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Journal of Proteome Research

Important Role of SUMOylation of Spliceosome Factors in Prostate Cancer Cells Donghua Wen#¶, Zhijian Xu#§, Li Xia#¶, Xinyi Liu¶, Yaoyao Tu¶, Hu Lei¶, Weiwei Wang¶, Tongdan Wang¶, Lili Song¶, Chunmin Ma¶, Hanzhang Xu¶, Weiliang Zhu§, Guoqiang Chen*¶, Yingli Wu*¶ ¶Department of Pathophysiology, Chemical Biology Division of Shanghai Universities E-Institutes, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. §Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ABSTRACT Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably-transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins

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of SUMO. Furthermore, we demonstrated the K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation. KEYWORDS: SUMOylation, SENP, SENP inhibitor, chemical proteomics, quantitative proteomics, SILAC, spliceosome, USP39 INTRODUCTION Prostate cancer is the most common type of cancer specific to men and is the second leading cause of cancer death affecting men in the western world:1 Up to 29 percent of men between 30 and 40 years of age and 64 percent of men between 60 and 70 years of age are diagnosed with small prostatic carcinomas.2 Although treatment at early stages with radiation therapy or prostatectomy is efficient in most cases, some patients develop a fatal hormone-refractory disease. The treatment options for these patients are limited to docetaxel-based chemotherapy, which extends survival just by a few months.3 Therefore, it is crucial to develop novel strategies to treat prostate cancer. To this end, revealing the mechanism underlying prostate cancer progression may provide potential targets for novel means of therapeutic interference. SUMOs are highly conserved 11 kDa proteins that are covalently attached to and detached from proteins to modulate their functions. Mammals cells express three

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major SUMO paralogues: SUMO1, SUMO2 and SUMO3. SUMOylation regulates many cellular processes, including protein-protein interaction, transcription, protein localization, cell cycle progression, DNA replication and repair, chromatin organization and RNA metabolism.4-6 SUMOylation is a highly dynamic and reversible process. Both the initial step to cleave the SUMO precursors into mature SUMOs and the deSUMOylation process to remove SUMOs from their substrate SUMOylated proteins are executed by Sentrin/SUMO-specific proteases (SENPs). Mammals have six SENPs: SENP1, SENP2, SENP3, SENP5, SENP6 and SENP7. They share a conserved catalytic domain that is typically found near their C-terminus but the N-termianl regions are largely unrelated.7 SENP1 and SENP2 localize to the nucleus and have endopeptiddase and isopeptidase activity for SUMO1, SUMO2 and SUMO3. By contrast, all other SENPs have a strong preference for SUMO2 and SUMO3. SENP3 and SENP5 localize in nucleoli, and catalyze SUMO2, SUMO3 processing and deconjugation. SENP6 and SENP7 localize within the nucleoplasm and are implicated in the editing of poly-SUMO chains.8-11 In addition, SENP1 and SENP2 have nuclear export sequences that allow these two SENPs to shuttle in and out of the nucleus.12 SENPs can be regulated by their localization, activity or through transcriptional control and post-translational regulation.7 The SENPs are critical for maintaining the level of SUMOylated and un-SUMOylated substrates required for normal physiology. Knockout of SENP1 or SENP2 in mice is embryonically lethal, indicating the importance to keep the balance between SUMOylation and deSUMOylation processes.13, 14 Recently, it has been reported that SENP1 and SENP3

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are elevated in prostate cancer tissues.12, 15 And overexpression of SENP1 correlates with the aggressiveness of the disease.16 Though SENP1 can transform normal prostate epithelia to a dysplastic state and directly modulate several oncogenic pathways in prostate cells, including androgen receptor, c-Jun, and cyclin D1,15, 17 other substrates of SUMO involved in the pathogenesis of prostate cancer are unknown. It remains difficult to assess globally the temporal aspects of SUMOylation due to the high activity of SENPs. In recent years, considerable research has been conducted to develop SENP inhibitors. However, the two kinds of SENP inhibitors available have low cell permeability, which limit their utilization in living cells.18-20 In this work, we identified a novel cell-permeable SENP inhibitor by high-throughput screening. Using this inhibitor to increase the abundance of SUMOylated proteins in cells and in combination with SILAC quantitative proteomics, we obtained the global SUMOylation profile of PC3 cells. Bioinformatic analysis revealed that the spliceosome pathway was highly enriched and several splicing factors such as USP39 were confirmed as novel SUMOylated proteins. We further identified the SUMOylation sites of USP39 and found that SUMO sites mutations of USP39 promoted the proliferation-enhancing effect of USP39 on both androgen-dependent and independent prostate cancer cells. This study revealed a novel role of spliceosome SUMOylation in the pathogenesis of prostate cancer and provides a lead compound for the development of isotype-specific SENP inhibitors and prostate cancer targeting agents. EXPERIMENTAL PROCEDURES

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Virtual Screening The crystal structure of SENP1 complexed with SUMO1 (PDB code: 2IY1)21 was retrieved from the protein data bank for molecular docking. C603A of SENP1 was mutated back to cysteine in Sybyl software package. The SPECS database contains approximately 200,000 small molecules (http:// www.specs.net/). About 100,000 molecules for virtual screening was generated with a druglike filter developed in our laboratory.22 The virtual screening was carried out on a 128-processor unix server. A hierarchical docking protocol with consensus scoring to evaluate the results of the virtual screening was used. DOCK4.023 was used as the primary screening tool against SENP1. Residues 94-98 of SUMO1, i.e., QTGGH, were chosen as ligand. SENP1 residues within 6 Å of ligand were isolated for the construction of the grid for docking screening. For DOCK4.0, the Kollman all-atom charges were assigned to the protein and the Gasteiger-Hückel charges were assigned to the small molecules. The top 25,000 molecules that were scored by DOCK4.0 were evaluated by CScore method implemented in Sybyl software package. The top 5,000 molecules that were rescored by CScore were reranked by AutoDock3.0.24 For AutoDock3.0 the polar hydrogen and Kollman United-atom charges were assigned to the protein while the Gasteiger-Hückel charges were assigned to the small molecules. The top 500 molecules were extracted and manually visualized for their binding modes and scaffold diversity. 117 candidate compounds from different scaffolds were purchased for the bioassay (Table S1). SUMO3-CHOP-Reporter DeSUMOylation Assay

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Effect

of

compounds

on

the

activity

of

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SENP1

was

evaluated

by

SUMO3-CHOP-Reporter DeSUMOylation Assay kit (LifeSensors Inc. Malvern, PA).25 20 nM SENP1 (LifeSensors) was preincubated with compounds for 10 min at 37 °C before the SUMO3-reporter-enzyme and the reporter substrate were added, followed by further incubation for 30 min. The fluorescence intensity was determined by the Synergy H4 Hybrid Microplate Reader with excitation at 475 nm and emission at 555 nm. The relative activity of SENP1 was determined by measuring the Relative Fluorescence Unit ( RFU ) value. Percentage inhibition (%) was calculated using the following equation [1-(RFU compound/RFU DMSO)] ×100. Plasmid Construction pBabe-Flag-SUMO1/SUMO2/SUMO3, pcDNA3.0-RGS-SENP1,

pBabe-Flag-SENP2/SENP3/SENP5,

pSAE1/SAE2/UBC9/SUMO2

and

pET28a

(His)-∆RanGAP1 are kind gift from Dr. Jinke Cheng (SJTU, Shanghai, China). Catalytic domain of SENP1 (amino acids 419-643) (SENP1C) was amplified from cDNA library of PC3 cells, and PCR product was cloned into the bacterial expression vector pET28a(+)-his. The UBC9 ORF was prepared by PCR using the pcDNA3.1-MYC-UBC9 plasmid (gift from Xiaowei Zhang, SJTU, Shanghai, China) as the template. pET28a(+)-his-UBC9 was constructed by inserting UBC9 ORF into pET28a(+)-his.

The

SUMO1

ORF

was

obtained

by

PCR

using

the

pBabe-Flag-SUMO1 plasmid as the template. pEGFP-C1-SUMO1 was made by cloning the SUMO1 ORF into pEGFP-C1. The SUMO2 ORF and SUMO2 ORF with the HA epitope at the 5’ end were amplified from the pBabe-Flag-SUMO2 plasmid.

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The SUMO2 ORF and HA-tagged SUMO2 were inserted into pET28a(+)-his and pBabe, respectively. The HSPA2 ORF was amplified by PCR from a cDNA library (from Jiahuai Han, Xiamen University, China). The USP39 ORF and HSPA1A ORF were prepared by PCR using pDEST_LTR_N_FLAG_HA_IRES_puro-USP39 (Addgene, No.19537) and pcDNA5/FRT/TO-HIS HSPA1A (Addgene, No.19446) as templates, respectively. The PCR products were subcloned into pBabe-3Flag vector. Truncation variants or SUMOylation sites mutants of USP39 were constructed by PCR using pBabe-3Flag-USP39 as template. All the constructs were confirmed to be correct by DNA sequencing. Purification of SENP1C and SUMO2-∆ ∆RanGAP1 pET28a(+)-SENP1C plasmid was transfected into E. coli BL21, and the expression of SENP1C was induced with 0.5 mM isopropyl-D-thiogalactoside (IPTG) at 16 °C for 12 h. pSAE1/SAE2/UBC9/SUMO2 and pET28a(+)-∆RanGAP1 (AA403-585 of Xenopus

laevis

RanGAP1)

were

co-transfected

into

E.coli

BL21.

SUMO2-∆RanGAP1 was induced with 1 mM IPTG at 25 °C for 12 h. Cell pellets were resuspended in lysis buffer (300 mM NaCl, 50 mM PBS pH 8.0, 10 mM imidazole, 10 mM β-mercaptoacetic ethanol and 10% glycerol) and sonicated. His-tagged proteins were purified using Ni-NTA-agarose and eluted with 250 mM imidazole in 300 mM NaCl, 50 mM PBS pH 8.0, 10 mM β-mercaptoacetic ethanol and 10% glycerol. In Vitro Gel-Based SENP Activity Assay SENP1C (5 nM) was incubated with compounds for 10 min at 37 °C in reaction

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buffer (50 mM Tris-HCl pH 8.0, 20 mM NaCl, 2 mM CaCl2, 2 mM DTT). Then SUMO2-∆RanGAP1 (3.92 µM, final concentration) was added and incubated for another 45 min at 37 °C. The reaction was terminated by adding loading buffer and boiling on heat block. The proteins were separated by 12% SDS-PAGE and visualized by coomassie brilliant blue (G250). Western Blotting Cells were washed with PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol). Cell lysates were centrifuged at 20,000g for 10 minutes, and proteins in the supernatants were quantified. Protein extracts were equally loaded to an 8% to 12% SDS–polyacrylamide gel, electrophoresed, and transferred to nitrocellulose membrane (Bio-Rad). The blots were stained with 0.2% Ponceau S red to ensure equal protein loading. After blocking with 5% nonfat milk in PBS, the membranes were probed with antibodies against Flag (Sigma-Aldrich), HA (Sigma-Aldrich), β-tubulin (Sigma-Aldrich), β-actin (Calbiochem), RGS.HisTM (Qiagen), His (Cell Signaling Technology). The signals were detected by chemiluminescence phototope-HRP kit (Cell Signaling) according to manufacturer’s instructions. As necessary, blots were stripped and reprobed with anti-β-actin or β-tubulin antibody as internal control. All experiments were repeated three times with the similar results. Proteasome Activity Assay More than 1×108 PC3 cells were collected, and lysed in digitonin buffer (50 mM Tris–HCl, pH 7.5, 250 mM Sucrose, 5 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.5 mM

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EDTA, 0.025% digitonin) on ice for 5 min. After centrifuging (20,000 g, 4 °C, 15 min), supernatant was collected for further centrifuging (300,000 g, 4 °C, 2 h). The precipitate was collected and redissolved in digitonin buffer. Protein concentration was determined by the Bradford assay. For proteasome activity assay, 5 µg extract product was added with 50 µM SI2 into reaction buffer (50 mM Tris–HCl, pH 7.5, 40 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5 mM ATP, 0.5 mg/ml BSA), and preincubated at 37 °C for 10 min. Then 20 mM substrate Suc-LLAY-AMC (Sigma-Aldrich)

for proteasomal chymotrypsin-like

activity),

for

Z-LLE-AMC

(Sigma-Aldrich)

caspase-like

activity,

and

BOC-LRR-AMC (Sigma-Aldrich) for proteasomal trypsin-like activity were added into the mixture, respectively and incubated at 37 °C for 60 min. Production of hydrolyzed 7-amido- 4-methyl-coumarin (AMC) groups was measured using the Synergy H4 Hybrid Microplate Reader VersaFluor Fluorometer with an excitation filter of 380 nm and an emission filter of 460 nm. The selective and irreversible proteasome inhibitor epoxomicin (10 µM) was used as a positive control for the inhibition of proteasome activity. Cathepsin B Activity Assay Effect of SI2 on the activity was evaluated by the cathepsin B activity assay kit (BioVision Inc, Mountain View, CA). 5×106 PC3 cells were collected and lysed in 350 µl lysis buffer for 10 min on ice. After centrifugation (15,000 rpm, 4 °C, 10 min), supernatant was pre-incubated with 50 µM E64 (inhibitor of cathepsin B) (Calbiochem), DMSO, 50 µM or 100 µM SI2 respectively at 37 °C for 30 min. Then

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substrate RR-AFC was added into the reaction systems, incubated for 1.5 h at 37 °C. The fluorescence of AFC was determined by the Synergy H4 Hybrid Microplate Reader with excitation at 400 nm and emission at 505 nm. Cathepsin D Activity Assay Effect of SI2 on the activity was evaluated by the cathepsin D activity assay kit (BioVision Inc). 1×106 PC3 cells were collected and lysed in 500 µl lysis buffer for 10 min on ice. After centrifuging (15,000 rpm, 4 °C, 10 min), supernatant was pre-incubated with 50 µM pepstatin A (inhibitor of cathepsin D), DMSO, 50 µM or 100 µM SI2 at 37 °C for 30 min. Then substrate GKPILFFRLK(Dnp)-D-R-NH2 labeled with 7-methoxycoumarin-4-ylacetyl (MCA) was added into the reaction systems, incubated for 1.5 h at 37 °C. The fluorescence of MCA was determined by the Synergy H4 Hybrid Microplate Reader with excitation at 328 nm and emission at 460 nm. Cell Culture and Generation of SUMO or USP39 Stably Transfected Cells HEK293T was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone), and PC3, LNCaP were grown in RPMI-1640 medium (Sigma-Aldrich) with 10% fetal bovine serum (FBS, Gibco BRL) in a 5% CO2/95% air humidified atmosphere at 37 °C. To produce virus, pGagpol and pVSVG (Clontec) were co-transfected with pBabe-Flag-SUMO1/2/3 or vector pBabe-Flag, respectively, into HEK293T cells using Fugene 6 (Roche Applied Science). Retrovirus-containing supernatant was harvested 48 h after transfection, filtered with 0.45 µM membrane (Millipore) to remove HEK293T Cells. Then retrovirus-containing supernatant was added into PC3

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cells with Polybrene (Sigma -Aldrich) to a final concentration of 8 µg/ml. 48 h later, puromycin (Sigma-Aldrich) was added into the medium. Positive polyclone cells were identified based on Flag-SUMO expression. Similarly, vector, Flag-USP39WT or Flag-USP39SM stably expressed PC3 and LNCaP cells were established. Stable Isotope Label, Immunoprecipitation and Protein Electrophoresis Stable isotope labeling was carried out using [12C6, lysine (referred to as 15

12

C

14

14

N4] arginine and [12C6,

N) for light medium, or [13C6,

15

14

N2]

N4] arginine and [13C6,

N2] lysine (referred to as 13C15N) (Cambridge Isotope Laboratories, Cambridge, MA)

for heavy medium. 12C14N-labeled and

13 15

C N-labeled PC3-Flag-SUMO cells were

treated with DMSO and 20 µM SI2 respectively for 1 h (SUMO1) or for 2 h (SUMO2 and SUMO3). The DMSO and SI2 treated cells were mixed in a 1:1 ratio (5×107 each), respectively. The mixture of treated cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P40, 1 mM EGTA, 1 mM Deoxycholic Acid, 20 mM N-Ethylmaleimide (Sigma-Aldrich), Roche complete EDTA-free protease inhibitor cocktail) on ice for 30 min, then sonicated. Lysates were cleared using centrifugation (12 000 rpm, 10 min, twice). After centrifuging, supernatant was interacted with anti-Flag antibody M2 beads (Sigma-Aldrich) at 4 °C for 6 h. Beads were washed more than 6 times with lysis buffer. Then the beads were boiled in loading buffer. Proteins were separated by 8% SDS-PAGE, stained with coomassie brilliant blue, and excised in slices, which were subjected to in-gel digestion. Gel slices were destained with 50% ACN in 50 mM ammonium bicarbonate. The dried gel were sequentially

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reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. The proteins were digested overnight at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI) at a protein-to-enzyme of 50:1. Peptides were extracted from gel slices with 60% ACN, 1% TFA, and then desalted with MicroTrap C8 (Michrom, US). LC-MS/MS Analysis The eluted peptides were lyophilized using a SpeedVac (ThermoSavant, USA), and resuspended in 20 µl 0.1% formic acid/2% acetonitrile. All mass spectrometric experiments were performed on a LTQ orbitrap “XL” mass spectrometer (Thermo Fisher Scientific, San Jose, CA) connected to a Paradigm MDLC nanoflow LC system (Michrom BioResources, USA) via an ADVANCE Spray Source LC-MS interface (Michrom Bioresources, USA). The peptides mixture were loaded onto a 15 cm with 0.1 mm inner diameter column packed with Magic C18AQ 3-µm Reversed Phase resins (Michrom BioResources, USA), and separated within a 120 min linear gradient from 100% solvent A (0.1% formic acid/2% acetonitrile/98% water) to 35% solvent B (0.1% formic acid/100% acetonitrile) at a flow rate of 500 nl/min. The spray voltage was set to 1.5 KV and the temperature of ion transfer capillary was 160 °C. The mass spectrometer were operated in positive ion mode and employed in the data-dependent mode to automatically switch between MS and MS/MS using the Tune and Xcalibur 2.5.5 software package. One full MS scan from 350 to 1800 m/z was acquired at high resolution R=100,000 (defined at m/z=400), followed by fragmentation of the ten most abundant multiply charged ions (singly charged ions and ions with unassigned charge states were excluded). Ions selected for MS/MS were limited on a dynamic

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exclusion list for 30 seconds. All MS/MS ion spectra were analyzed using Sequest26 (Thermo Fisher Scientific, San Jose, CA, USA; version v.27, rev. 11) which were incorporated into the Sorcerer engine version 4.0.4 build (Sage-N Research). Sequest were set up to search the ipi.HUMAN.v3.87 database (91464 entries) (ftp.ebi.ac.uk/pub/databases/IPI/current) with its target-decoy database as a reversed complement assuming the semi-enzyme tryptic digestion allowed for three missed tryptic cleavages with full mass from 600 to 4600. The precursor ion tolerance was set to ±10 p.p.m and MS2 ions tolerance was set to 1 Da. Searches parameters for total peptides were allowed for a static modification of carbamidomethyl C (+57.021465) on cysteine, and dynamic modifications of oxidized M (+15.99492) on methionine, NEM (+125.047679) on cysteine, SILAC-labels K (6C2N, +8.014199) on lysine and SILAC-labels R (6C4N, +10.008269) on arginine with up to 4 total dynamic modifications, up to 3 of any particular dynamic modification. DATSelect 2.0.39 was used to filter and group the outcome derived from SORCERER-SEQUEST. The cutoff values of filter threshold were placed on XCorr 2+, 3+, 4+: > 2.5, > 3.0, > 3.5 and DeltaCN > 0.08, two peptides required for total peptides to protein identification with estimated false-discovery rates (FDR) < 1%. Each raw data converted by RAWXreact 1.9.9.1 combined with its matching output file filtered by DATSelect were processed by Census 1.72 to generate final fold-change ratios in peptide amounts for each time point. In brief, based on the results of peptides identification, either experimental heavy or light variants of each peptide was preset and used to calculate and identify

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the theoretical heavy or light ion peaks in the high mass accuracy of tolerance window of ± 10 p.p.m. The intensity of the peaks was used to construct ion chromatograms. A minimum of 1.3 ratio counts are required to perform protein quantitation. In Vitro SUMOylation Assay Flag-USP39, Flag-HSPA1A or Flag-HSPA2 was immunoprecipitated by anti-Flag M2 beads (Sigma-Aldrich) from transfected HEK293T cells. Then Flag-tagged substrates were competitively eluted by 3×Flag peptide (Sigma-Aldrich) from the anti-Flag M2 beads. Five microliter eluent were incubated with SAE1/SAE2 (100 nM, Bostonbiochem), UBC9 (2 µM) and SUMO1/2/3 (32 µM) in reaction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM ATP, 2 mM MgCl2) at 37 °C for 2 h. The reaction was terminated and immunoblots were probed with anti-Flag or anti-His antibody. Cell Proliferation Assay Wild type or SUMO sites mutant USP39 stably transfected PC3 or LNCaP cells were cultured in 96-well plates in 200 µl of media, respectively. At different time point, cell proliferation was evaluated by the CCK-8 assay (WST-8; Cell Counting Kit-8 from Dojindo, Kumamoto, Japan). Briefly, each well were pulsed by addition of 10 µl of WST8 for 3 h. WST-8 is converted to WST-8-formazan upon bioreduction in the presence of the electron carrier 1-methoxy-5-methylphenaziniummethyl sulfate that is abundant in viable cells. Absorbance readings at a wavelength of 450 nm were taken on Synergy H4 Hybrid Microplate Reader. The optic density (OD) values of day 1, 2, 4, 6 were normalized by dividing by the OD values of 10 h, when the cells adhere to

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the bottom of the well. Bioinformatics Analysis The DAVID web server was used for KEGG pathway analysis of the identified proteins. When analyzing the common enriched functional categories among the identified proteins, all genes in the genome were used as the background population; when analyzing each cluster of the identified proteins, all the identified proteins were used as the background population. Statistical Analysis Student’s t-test was used to evaluate the difference between two different treatments. A p value of less than 0.05 was considered statistically significant. RESULTS SI2 Is a SENP-Specific Inhibitor From the virtual screening (see EXPERIMENTAL PROCEDURES, Figure S1), 117 candidate compounds (Table S1) from different scaffolds were selected and their potency for SENP1 inhibition was analyzed using the SUMO3-CHOP-Reporter DeSUMOylation Assay kit at 50 µM. Among them, SI2 (Figure 1A) showed the most potent inhibition for SENP1 activity and was selected for further investigation. To confirm the inhibitory effect of SI2 on SENP1, an in vitro gel-based SENP activity assay was performed. As shown in Figure 1B, 50 µM SI2 markedly inhibited SENP1C-mediated cleavage of ∆RanGAP1-SUMO2, while SI30, which had no inhibitory activity on SENP1 as examined by the DeSUMOylation Assay kit, could not inhibit SENP1C-induced cleavage of ∆RanGAP1-SUMO2 (Figure 1B). The IC50

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of SI2 on SENP1C inhibition was determined as 1.29 µM (Figure 1C and Figure S2). Docking study (Figure 1D) showed that SI2 resided in a tunnel within SENP1, which prevented the binding of SUMO1 to SENP1. Notably, the chlorobenzene moiety of SI2 occupied the space of the Gly-Gly motif at the C terminus of SUMO1. There were several favorable interactions between SI2 and SENP1, i.e., π−π interaction between the chlorobenzene moiety and W465, π−π interaction between the biphenyl group and F496, π−π interaction between the biphenyl group and H529, and hydrogen bonding between the carbonyl group and H529. As SENPs belong to the family of cysteine proteases, we investigated the effects of SI2 on several other proteases of this family. Even at 100 µM, SI2 had little effect on cysteine proteases such as cathepsin B and cathepsin D (Figure 1E and 1F) while E64 and pepstatin A inhibited the activity of cathepsin B and cathepsin D, respectively. In addition, it has been reported that treatment of Hela cells with the proteasome inhibitor MG132 led to the accumulation of SUMOylated proteins.27 We also tested the effect of SI2 on the proteasome activity in vitro. Unlike the positive control Epoximicin, 50 µM SI2 had little inhibitory effect on chymotrypsin-like, trypsin-like, and caspase-like activity of proteasome (Figure. 1G). These results indicate that SI2 can specifically inhibit SENP1 activity. As SENP1C contained only the catalytic domain, we sought to determine whether SI2 inhibited the activity of full-length SENP1 in cells. To this end, HEK293T cells were transfected with full-length SENP1 and Flag-tagged SUMO2 and were then treated with SI2. As shown in Figure 1H, with increasing concentrations of SI2, more

16


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SUMO-modified proteins accumulated, indicating that SI2 can inhibit the isopeptidase activity of the full-length SENP1 in cells. Since SI2 can inhibit the activity of SENP1 and the catalytic domain is conserved among different SENP proteins, we next asked whether SI2 affected the activity of other SENPs. HEK293T cells were transfected with HA-SUMO2 and full-length Flag-tagged SENP2, 3, or 5 and the cells were then treated with SI2. As shown in Figure S3A-C, transfection of SENP2, 3, and 5 all caused the disappearance of the high molecular weight smears representing SUMOylated proteins. SI2 treatment could reverse the deSUMOylation activity of SENP2 and SENP3 (Figure S3A and S3B); however, SI2 could not inhibit SENP5-induced deSUMOylation (Figure S3C). These results indicate that SI2 is a relative specific inhibitor of SENPs. SI2 Increases the Amount of SUMOylated Proteins in Prostate Cancer Cells Given that SI2 can inhibit the activity of the exogenous SENPs, we next investigated whether SI2 could inhibit the activity of endogenous SENPs. Elevated expression of SENP1 and SENP3 has been observed in prostate cancer cells and is associated with the progression of prostate cancer.15-17 We therefore chose PC3, an androgen receptor negative prostate cancer cell line with high expression of SENP1, to evaluate the possible effect of SI2 on SENPs activity. SI2 treatment could increase the endogenous SUMOylated proteins without obviously changing the protein level of unique conjugating enzyme UBC9, SENP1/2/3 (Figure S4). Because the overall intracellular concentration of SUMO1 is smaller and it is less dynamic, and there are no specific antibodies to distinguish endogenous SUMO2 from SUMO3, we transfected the PC3

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Page 18 of 47

cells stably with pBabe-Flag-SUMO1/2/3 plasmids, respectively, to increase the pool of free SUMO1 and to distinguish SUMO2 or SUMO3 modified proteins. SI2 treatment

induced

massive

increase

of

the

SUMOylated

proteins

in

SUMO2/3-transfected PC3 cells and moderate increase in SUMO1-transfected PC3 cells, as reflected by the appearance of the smear bands at high molecular weights (Figure 2A-C). These results suggest that SI2 can inhibit the isopeptidase activity of endogenous SENPs and subsequently lead to the accumulation of SUMOylated proteins. Quantitative Proteomics Strategy to Identify SUMOylated Proteins Having demonstrated that SI2 is a SENP inhibitor, we next used this compound as a chemical tool in stable isotope labeling with amino acids (SILAC) quantitative proteomics to enrich and identify SUMOylated proteins in prostate cancer cells (Figure 3A). PC3 cells stably expressing Flag-SUMO1 were labeled with either 12 14

C N

or

13 15

C N

(see

EXPERIMENTAL PROCEDURES).

Flag-SUMO1 PC3 cells were treated with DMSO and

12 14

C N-labeled

13 15

C N-labeled Flag-SUMO1

PC3 cells were treated with 20 µM SI2. As expected, compared with DMSO-treated cells (DMSOL), SI2-treated cells (SI2H) showed an increase of SUMO target conjugation (Figure 3B, first and second lane). The extracts from DMSOL and SI2H were then mixed in a 1:1 ratio and Flag-tagged proteins were pulled down by immunoprecipitation and detected with anti-Flag antibody by Western blotting. The results showed that the SUMOylated proteins were enriched in the immunoprecipitate (Figure 3B, third lane). Finally, the immunoprecipitate was separated by SDS-PAGE

18


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and the coomassie-stained gel was cut (Figure 3E). Proteins were in gel digested by trypsin. The resulting peptide mixture was analyzed by LTQ-Orbitrap hybrid mass spectrometer. Same procedures were repeated for PC3 cells stably expressing Flag-SUMO2 or Flag-SUMO3 (Figure 3C-D and 3F-G). Although the SILAC technique can quantify changes less than 10%,28 we chose 1.3 fold as a conservative cutoff ratio to detect SENP-regulated proteins, which may play a role in SENP-mediated prostate cancer development (Table S2 and Figure S5). Among more than 900 identified proteins (Table S3-5), 231 proteins increased ≥1.3 fold, including 39 SUMO1-conjugated proteins, 145 SUMO2-conjugated proteins, and 99 SUMO3- conjugated proteins (Table S3-6). Of the 231 putative target proteins of SUMO, 24 proteins are known SUMO target proteins (Table S6). These results demonstrate that the strategy to identify the endogenous SUMOylated protein is effective. Spliceosome Factors USP39, HSP1A1, HSPA2 Are Novel SUMO Target Proteins To determine their possible functional roles, the 231 putative SUMO targeted proteins (Table S6) were subjected to bioinformatic analysis. From the KEGG pathway analysis, the spliceosome pathway was highly enriched (Table I). Among the 20 putative SUMO-targeted proteins, HNRNPK, HNRNPM, DDX5 have been reported as SUMO targets. 29-31 To determine whether other factors are SUMO substrates, we performed the in vitro SUMOylation assay. As USP39 was a putative SUMO2 and SUMO3 modified protein (Tables S4-6) and is the ortholog of yeast spliceosomal Sad1p, we first selected USP39 for further analysis. As shown in Figure 4A, slow

19



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migration bands appeared after Flag-tagged USP39 reacted with SAE1/SAE2, His-UBC9, and SUMO2. No such band could be seen when the reactions were performed in the absence of SAE1/SAE2/UBC9/SUMO2. Furthermore, USP39 could also be modified by SUMO3 (Figure 4B) and SUMO1（Figure 4C）. These results indicate that USP39 may not have preference to SUMO paralogues in vitro. Using immunofluorescence staining, we further demonstrated that SUMO1 and SUMO2/3 could co-localize with USP39 (Figure S6). Similarly, we demonstrated that HSPA1A (a putative SUMO1-3 modified protein, Tables S3-6) and HSPA2 (a putative SUMO2-3 modified protein, Table S4-6), the other two splicing factors, could also be modified by SUMO1 (Figure. 4D and E). Of note, all the above SUMOylated proteins had a molecular shift higher than 11 kDa (1 SUMO). There are two possibilities to explain this: one SUMO molecule conjugated on several lysines (multiSUMOylation) or the presence of a growing polymeric SUMO chain on a single lysine (polySUMOylation).32 K6, K16, K29, K51 and K73 Are the SUMO Acceptor Sites of USP39 As USP39 has a vital role in assembling functional spliceosomes, we next attempted to find its SUMO acceptor sites. As shown in Figure 4, USP39 had several shift bands higher than 11 kDa in the in vitro SUMOylation assay. This means USP39 may have multiple SUMO sites or the formation of polychains. There are 45 lysines in USP39. Thus, it is not easy to figure out which lysines are possible SUMO sites for USP39, as SUMOylation can occur at both the SUMO-consensus motif and non-consensus motif. In order to narrow down the searching region of SUMO sites, we determined to

20


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construct truncations of USP39 for the in vitro SUMOylation assay. USP39 contains mainly four domains: Arginine-serine-rich (RS)-like domain, ZnF domain, UCH1 domain, and UCH2 domain33 (Figure 5A). The RS domain has been shown to be essential for the maturation of spliceosome.34 Therefore, we first constructed two truncated forms of USP39, one with amino acids 1-100 (containing the RS-like domain, AA1-100, RSWT), and the other with amino acids 101-565 (containing the ZnF, UCH1, and UCH2 domains, AA101-565) and transfected them into HEK293T cells (Figure 5A). In vitro SUMOylation assay was performed with the purified Flag-tagged truncated USP39 proteins (Figure 5B). As shown in Figure 5C, obvious SUMOylated bands were observed in the truncation containing only the RS-like domain, but not in the truncation without the RS-like domain. These data suggest that the SUMOylation sites of USP39 are located in RS-like domain. There are 6 lysines in the RS-like domain (Figure 6A). To ascertain the SUMOylation sites, we constructed 6 mutants from the full-length USP39, namely USP39-RSK6, -RSK16, -RSK29, -RSK51, -RSK73, -RSK91. In each mutant, the mutation was performed only in the RS-like domain (amino acids 1-100) and 5 out of 6 lysine residues were mutated into arginines. For example, in USP39-RSK6 mutant, all lysines except for lysine 6 were mutated into arginines in the RS-like domain. In vitro SUMOylation assay was performed with the wild type USP39 (USP39-RSWT) and the 6 purified Flag-tagged mutants. As shown in Figure 6B and C, obvious SUMOylated bands were observed in the USP39-RSK29, -RSK51, -RSK73 mutants and weaker bands were observed in the USP39-RSK6, -RSK16 mutants. However, complete loss of

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Page 22 of 47

SUMOylation was observed in the USP39-RSK91 mutants. These data suggest that the K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. To test whether the SUMO sites identified are applicable to cellular conditions, we transfected the full-length USP39 (Flag-USP39WT) and the SUMO sites mutant (all 6 lysines but the K91) USP39 (Flag-USP39SM) into PC3 cells in the presence of exogenous GFP-SUMO1. Flag-USP39WT and Flag-USP39SM were immunoprecipitated and subjected to western blotting using the anti-Flag and anti-SUMO antibodies. As expected, several slower migrating bands were observed in USP39WT but not in USP39SM immunoprecipitate (Figure 6D). These results demonstrate that K29, K51, and K73, and to a lesser extent, K6, K16, are the SUMO acceptor sites of USP39. SUMOylation of USP39 Affects Proliferation of Prostate Cancer Cells To address the functional consequences of SUMO modification on USP39, the androgen-independent prostate cancer cells PC3 were stably transfected with Flag-USP39WT or Flag-USP39SM (Figure 7A). The proliferation of the transfected cells were monitored by the CCK-8 assay. As shown in Figure 7B, on day 6, the number of Flag-USP39WT transfected PC3 cells were significantly more than vector-transfected cells (P < 0.05). Intriguingly, Flag-USP39SM-transfected PC3 cells grew more rapidly than Flag-USP39WT-transfected PC3 cells (P < 0.05). Similar phenomena were observed in the androgen-dependent prostate cancer cells LNCaP (Figure 7C-D). These results indicate that deSUMOylated USP39 has stronger proliferation

enhancing

effect

than

USP39

in

androgen-dependent

androgen-independent prostate cancer cells.

22


and

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DISCUSSION Although the importance of SENP1 in the pathogenesis of prostate cancer has been well demonstrated, the SUMOylated proteins affected by SENP1 are largely unknown. In this study, using a chemical proteomics approach, we identified candidate SUMOylated proteins regulated by SENPs in prostate cancer cells, in which the spliceosome factors are highly enriched. We further provided evidence that the SUMOylation of USP39, a bona fide spliceosome factor, is involved in the proliferation regulation of prostate cancer cells. Due to the high activity of SENPs in prostate cancer cells, it is difficult to identify the SUMOylated proteins involved in the pathogenesis, development and aggressiveness of prostate cancer. One approach to overcome this problem is to inhibit the SENPs activities by SENP inhibitors, which may in turn induce the accumulation of SUMOylated proteins. To date, two kinds of covalent inhibitors have been reported, the peptide vinyl sulfones inhibitor and the azaepoxide and acyloxymethyl ketone (AOMK) inhibitor.18-20 Both of them are designed based on the SUMO sequence modified at the C terminus with electrophilic entities capable of reacting with the thiol group of the active-site cysteine. However, due to their peptide character, these inhibitors have poor membrane permeability. In this study, we identified SI2 as a new cell-permeable SENP-specific inhibitor, which was supported by the following evidence: (a) The molecular docking analysis indicated SI2 resided in a tunnel within SENP1, which prevented the binding of SUMO1 to SENP1; (b) both the gel-based and the SUMO-CHOP-reporter in vitro assays demonstrated the

23



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Page 24 of 47

SENP1-inhibiting activity of SI2; (c) SI2 treatment caused the accumulation of SUMOylated proteins in PC3 cells, reflecting the inhibition of SENP activity in cells. Compared with the above two peptide-containing SENP inhibitors, SI2 is equal or more potent and avoids the disadvantageous effect of peptides on cell permeability.18-20 Therefore, SI2 represents a novel class of SENP inhibitor and a good chemical tool to enrich global SUMOylated proteins in cells, especially in the prostate cancer cells with overexpressed SENP1 and SENP3. However, except SENP5, the selectivity of SI2 against SENP1, SENP2 and SENP3 is so far unsatisfactory. Using SI2 as a lead compound to develop novel specific inhibitors for each SENP would be an important subject for further investigation. Using more sensitive methodologies is another approach to increase the chance of identifying SUMOylated proteins. SILAC is a sensitive and reliable quantitative proteomics method that can quantify changes smaller than 10%.28

In this work, in

order to set threshold for the regulated proteins, we analyzed the H/L ratio of identified peptides with determinant scores (R2) > 0.9 in the PC3Flag-SUMO1, PC3Flag-SUMO2 and PC3Flag-SUMO3 cells, respectively. As shown in Figure S5, the distribution of all peptides between ±1.3 fold was about 95.0% for PC3Flag-SUMO1, 95.7% for PC3Flag-SUMO2 and 95.1% for PC3Flag-SUMO3. Therefore, these results may provide a statistical basis for the selection of a fold change ratio of 1.3.

27

Under this

condition, 24 known SUMOylation substrates were identified (Table S6), including several novel SUMOylation substrates such as USP39, HSP1A1, and HSPA2, indicating the validity of the cutoff value used. It is interesting to note that SUMO2

24


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was found in SUMO1 immunoprecipitate (Table S3) and all three paralogues were present in SUMO2 and SUMO3 immunoprecipitate. As SUMO2 and SUMO3 can form conjugated chains through a single conserved acceptor lysine, and SUMO1 may terminate chains that are elongated through serial conjugation of SUMO2 and SUMO3, it is therefore reasonable to see all three paralogues were present in SUMO2 and SUMO3 immunoprecipitate. It is more interesting to see SUMO2 was found in SUMO1 immunoprecipitate. Despite SUMO1 is not equivalent in conserved acceptor lysine to SUMO2 and SUMO3, it can form poly-SUMO1 chains.32 Thus, one possible explanation for this is that SUMO2 but not SUMO3 can also conjugate to SUMO1. However, further investigations are needed to demonstrate this hypothesis. Spliceosome is an intracellular protein/RNA complex that guides pre-mRNA splicing in eukaryotic cells and contains over 150 distinct proteins and 5 small nuclear (sn)

RNAs.35

Post-translational

modifications

such

as

phosphorylation,

ubiquitination, acetylation, and other modifications have been shown to contribute to splicing regulations involving the physical rearrangements of the spliceosome, the timing of these rearrangements, the activities of splicing factors, or the mRNP composition assembled in a particular situation.36 However, whether SUMOylation plays an important role in this process has not been shown. In this study, we demonstrated that splicing factors including USP39, HSP1A1, and HSPA2 are novel SUMO substrates. USP39 is essential for U4/U6 biogenesis and the recruitment of the tri-snRNP to the pre-spliceosome to assemble the mature spliceosome. USP39 contains a central zinc finger and 2 ubiquitin C-terminal hydrolase (UCH) domains.

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Page 26 of 47

The N-terminal domain is rich in arginines, serines, and glutamic acids and resembles the RS domain of serine/arginine (SR)-related proteins, such as SNRP70 and RY1.33 The RS domain in SR proteins is essential to recruit the tri-snRNP to the pre-spliceosome.34 Interestingly, we demonstrate that all 5 SUMOylation sites of USP39 are located in the RS-like domain, indicating that SUMOylation of USP39 may affect the recruitment of tri-snRNP. Spliceosome factors have been shown to be associated with cancer development. For example, SF3B1 (Splicing factor 3B subunit 1) is highly mutated in various hematological

malignancies

myelodysplastic syndromes.37,

such 38

as

chronic

lymphocytic

leukemia

and

SAM68 (Src associated in mitosis; 68 kDa), an

RNA-binding protein involved in several aspects of mRNA processing, is frequently up-regulated in human prostate cancer cells. It can enhance splicing of cyclin D1 and CD44. Down-regulation of its expression or activity affects prostate cancer cell proliferation, motility and survival. Interestingly, SAM68 is a SUMO substrate. SUMOylated SAM68 acts as a transcriptional inhibitor of cyclin D1 expression.39, 40 More recently, Wang et al. demonstrated that USP39 are expressed at a higher level in breast cancer cells and knockdown of USP39 markedly reduces the proliferative and colony-forming ability of MCF-7 cells.41 Consistent with this report, we demonstrated that the overexpression of USP39 could enhance the proliferation of the androgen-independent PC3 and androgen-dependent LNCaP prostate cancer cells, suggesting that USP39 may also play a role in prostate cancer development. Intriguingly, mutation of the SUMOylation sites of USP39 further strengthened its

26


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ability to enhance the proliferation of prostate cancer cells. These data indicate SUMO modifications have a great impact on the function of USP39 and deSUMOylation of USP39 by SENPs may contribute to SENP-mediated prostate cancer development. Despite USP39 is mainly a nuclear protein, it distributes in both nuclear and cytoplasm. And it is co-localized with SUMO1 in nuclear and SUMO2/3 in both nuclear and cytoplasm (Figure S6). However, both SENP1 and SENP2 had partially co-localization with USP39 (Figure S6). Whether deSUMOylation of USP39 could change its localization, interaction with other proteins or stability and whether the effect of deSUMOylation of USP39 is mediated by the abnormal recruitment of tri-snRNP are currently unknown. CONCLUSIONS Using a chemical proteomics approach, we analyzed the SUMOproteome in prostate cancer cells and revealed that SUMOylation of the spliceosome factors might play an important role in the pathogenesis of prostate cancer. We provided evidence that USP39, a bona fide splicing factor, could be modified by SUMOylation and deSUMOlyation of USP39 strengthened its proliferation-enhancing effect in prostate cancer cells. Further investigation on how SUMO modification affects its function may provide new insights in prostate cancer progresses and new therapeutic intervention targets. In addition, optimization of SI2 may lead to novel isotype-specific inhibitors for SENPs and prostate cancer targeting agents. ASSOCIATED CONTENT Supporting Information

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Page 28 of 47

Figure S1. Virtual screening procedures for discovering SENP1 inhibitors. Figure S2. SI2 inhibits SENP1 activity in a dose-dependent manner in vitro. Figure S3. SI2 inhibits the activity of SENP2 and SENP3 in cells. Figure S4. Effect of SI2 on endogenous SUMOs, UBC9 and SENPs in PC3 cells. Figure S5. Distribution of log2 ratios of detected peptides in labeled untreated SUMO1/2/3 transfected PC3 cells. Figure S6. Co-localization assay of SUMO1, SUMO2/3, SENP1 and SENP2 with USP39 in PC3 cells. Table S1. List of compound structures and inhibition ratio. Table S2. List of detected peptides of labeled untreated SUMO1-3 transfected PC3 cells. Table S3. List of proteins identified from SI2-treated PC3 cells stably expressed Flag-SUMO1. Table S4. List of proteins identified from SI2-treated PC3 cells stably expressed Flag-SUMO2. Table S5. List of proteins identified from SI2-treated PC3 cells stably expressed Flag-SUMO3. Table S6. List of proteins for pathway analysis This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION *Corresponding Author Yingli

Wu,

Tel:

[email protected].

86-21-63846590-776916. Guoqiang

Chen,

Tel:

Fax:

86-21-64154900.

E-mail:

86-21-63846590-776529.

28


Fax:

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86-21-64154900. E-mail: [email protected] Author Contributions #

These authors contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We thank Dr. Han-Yi Zhuang for her reading and editing. This work was supported in part by grants from National Basic Research Program of China (973 Program) (NO. 2010CB912104, 2009CB149104), National Natural Science Foundation of China (91013008, 91313303, 31100980, 81272886), Science and Technology Committee of Shanghai (11JC1406500), the Ministry of Science and Technology (2012AA01A305), SMC Program of Shanghai Jiao Tong University. REFERENCES (1). Siegel, R.; Naishadham, D.; Jemal, A., Cancer statistics, 2012. CA. Cancer J. Clin. 2012, 62, (1), 10-29. (2). Nelson, W. G.; De Marzo, A. M.; Isaacs, W. B., Prostate cancer. N. Engl. J. Med. 2003, 349, (4), 366-81. (3). Sartor, A. O., Progression of metastatic castrate-resistant prostate cancer: impact of therapeutic intervention in the post-docetaxel space. J. Hematol. Oncol. 2011, 4, 18. (4). Johnson, E. S., Protein modification by SUMO. Annu. Rev. Biochem. 2004, 73, 355-82.

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(5). Yeh, E. T., SUMOylation and De-SUMOylation: wrestling with life's processes. J. Biol. Chem. 2009, 284, (13), 8223-7. (6). Nie, M.; Xie, Y.; Loo, J. A.; Courey, A. J., Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation. PLoS One 2009, 4, (6), e5905. (7). Hickey, C. M.; Wilson, N. R.; Hochstrasser, M., Function and regulation of SUMO proteases. Nat. Rev. Mol. Cell. Biol. 2012, 13, (12), 755-66. (8). Mukhopadhyay, D.; Ayaydin, F.; Kolli, N.; Tan, S. H.; Anan, T.; Kametaka, A.; Azuma, Y.; Wilkinson, K. D.; Dasso, M., SUSP1 antagonizes formation of highly SUMO2/3-conjugated species. J. Cell Biol. 2006, 174, (7), 939-49. (9). Shen, L. N.; Geoffroy, M. C.; Jaffray, E. G.; Hay, R. T., Characterization of SENP7, a SUMO-2/3-specific isopeptidase. Biochem. J. 2009, 421, (2), 223-30. (10). Citro, S.; Chiocca, S., Sumo paralogs: redundancy and divergencies. Front Biosci. (Schol. Ed.) 2013, 5, 544-53. (11). Watts, F. Z., Starting and stopping SUMOylation. What regulates the regulator? Chromosoma 2013, 122, (6), 451-63. (12). Bawa-Khalfe, T.; Yeh, E. T., SUMO Losing Balance: SUMO Proteases Disrupt SUMO Homeostasis to Facilitate Cancer Development and Progression. Genes Cancer 2010, 1, (7), 748-52. (13). Cheng, J.; Kang, X.; Zhang, S.; Yeh, E. T., SUMO-specific protease 1 is essential for stabilization of HIF1alpha during hypoxia. Cell 2007, 131, (3), 584-95. (14). Kang, X.; Qi, Y.; Zuo, Y.; Wang, Q.; Zou, Y.; Schwartz, R. J.; Cheng, J.; Yeh, E.

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T., SUMO-specific protease 2 is essential for suppression of polycomb group protein-mediated gene silencing during embryonic development. Mol. Cell 2010, 38, (2), 191-201. (15). Cheng, J.; Bawa, T.; Lee, P.; Gong, L.; Yeh, E. T., Role of desumoylation in the development of prostate cancer. Neoplasia 2006, 8, (8), 667-76. (16). Wang, Q.; Xia, N.; Li, T.; Xu, Y.; Zou, Y.; Zuo, Y.; Fan, Q.; Bawa-Khalfe, T.; Yeh, E. T.; Cheng, J., SUMO-specific protease 1 promotes prostate cancer progression and metastasis. Oncogene 2013, 32, (19), 2493-8. (17). Bawa-Khalfe, T.; Cheng, J.; Lin, S. H.; Ittmann, M. M.; Yeh, E. T., SENP1 induces prostatic intraepithelial neoplasia through multiple mechanisms. J. Biol. Chem. 2010, 285, (33), 25859-66. (18). Hemelaar, J.; Borodovsky, A.; Kessler, B. M.; Reverter, D.; Cook, J.; Kolli, N.; Gan-Erdene, T.; Wilkinson, K. D.; Gill, G.; Lima, C. D.; Ploegh, H. L.; Ovaa, H., Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol. Cell Biol. 2004, 24, (1), 84-95. (19). Borodovsky, A.; Ovaa, H.; Meester, W. J.; Venanzi, E. S.; Bogyo, M. S.; Hekking, B. G.; Ploegh, H. L.; Kessler, B. M.; Overkleeft, H. S., Small-molecule inhibitors

and

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and

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proteases.

Chembiochem. 2005, 6, (2), 287-91. (20). Albrow, V. E.; Ponder, E. L.; Fasci, D.; Bekes, M.; Deu, E.; Salvesen, G. S.; Bogyo, M., Development of small molecule inhibitors and probes of human SUMO deconjugating proteases. Chem. Biol. 2011, 18, (6), 722-32.

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(21). Shen, L.; Tatham, M. H.; Dong, C.; Zagorska, A.; Naismith, J. H.; Hay, R. T., SUMO protease SENP1 induces isomerization of the scissile peptide bond. Nat. Struct. Mol. Biol. 2006, 13, (12), 1069-77. (22). Zheng, S.; Luo, X.; Chen, G.; Zhu, W.; Shen, J.; Chen, K.; Jiang, H., A new rapid and effective chemistry space filter in recognizing a druglike database. J. Chem. Inf. Model. 2005, 45, (4), 856-62. (23). Ewing, T. J. A.; Kuntz, I. D., Critical evaluation of search algorithms for automated molecular docking and database screening. Journal of Computational Chemistry 1997, 18, (9), 1175-89. (24). Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J., Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. Journal of Computational Chemistry 1998, 19, (14), 1639-62. (25). Leach, C. A.; Tian, X.; Mattern, M. R.; Nicholson, B., Detection and characterization of SUMO protease activity using a sensitive enzyme-based reporter assay. Methods Mol. Biol. 2009, 497, 269-81. (26). Yates, J. R., 3rd; Eng, J. K.; McCormack, A. L.; Schieltz, D., Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 1995, 67, (8), 1426-36. (27). Matafora, V.; D'Amato, A.; Mori, S.; Blasi, F.; Bachi, A., Proteomics analysis of nucleolar SUMO-1 target proteins upon proteasome inhibition. Mol. Cell Proteomics 2009, 8, (10), 2243-55.

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(28). Ong, S. E.; Kratchmarova, I.; Mann, M., Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J. Proteome Res. 2003, 2, (2), 173-81. (29). Li, T.; Evdokimov, E.; Shen, R. F.; Chao, C. C.; Tekle, E.; Wang, T.; Stadtman, E. R.; Yang, D. C.; Chock, P. B., Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, (23), 8551-6. (30). Vertegaal, A. C.; Ogg, S. C.; Jaffray, E.; Rodriguez, M. S.; Hay, R. T.; Andersen, J. S.; Mann, M.; Lamond, A. I., A proteomic study of SUMO-2 target proteins. J. Biol. Chem. 2004, 279, (32), 33791-8. (31). Fuller-Pace, F. V.; Jacobs, A. M.; Nicol, S. M., Modulation of transcriptional activity of the DEAD-box family of RNA helicases, p68 (Ddx5) and DP103 (Ddx20), by SUMO modification. Biochem. Soc. Trans. 2007, 35, (Pt 6), 1427-9. (32). Yang, M.; Hsu, C. T.; Ting, C. Y.; Liu, L. F.; Hwang, J., Assembly of a polymeric chain of SUMO1 on human topoisomerase I in vitro. J. Biol. Chem. 2006, 281, (12), 8264-74. (33). Makarova, O. V.; Makarov, E. M.; Luhrmann, R., The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes. EMBO J. 2001, 20, (10), 2553-63. (34). Roscigno, R. F.; Garcia-Blanco, M. A., SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome. RNA 1995, 1, (7), 692-706. (35). Wahl, M. C.; Will, C. L.; Luhrmann, R., The spliceosome: design principles of a

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dynamic RNP machine. Cell 2009, 136, (4), 701-18. (36). McKay, S. L.; Johnson, T. L., A bird's-eye view of post-translational modifications in the spliceosome and their roles in spliceosome dynamics. Mol. Biosyst. 2010, 6, (11), 2093-102. (37). Wang, L.; Lawrence, M. S.; Wan, Y.; Stojanov, P.; Sougnez, C.; Stevenson, K.; Werner, L.; Sivachenko, A.; DeLuca, D. S.; Zhang, L.; Zhang, W.; Vartanov, A. R.; Fernandes, S. M.; Goldstein, N. R.; Folco, E. G.; Cibulskis, K.; Tesar, B.; Sievers, Q. L.; Shefler, E.; Gabriel, S.; Hacohen, N.; Reed, R.; Meyerson, M.; Golub, T. R.; Lander, E. S.; Neuberg, D.; Brown, J. R.; Getz, G.; Wu, C. J., SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N. Engl. J. Med. 2011, 365, (26), 2497-506. (38). Cazzola, M.; Rossi, M.; Malcovati, L., Biologic and clinical significance of somatic mutations of SF3B1 in myeloid and lymphoid neoplasms. Blood 2013, 121, (2), 260-9. (39). Cappellari, M.; Bielli, P.; Paronetto, M. P.; Ciccosanti, F.; Fimia, G. M.; Saarikettu, J.; Silvennoinen, O.; Sette, C., The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells. Oncogene 2013, [Epub ahead of print]. (40). Babic, I.; Cherry, E.; Fujita, D. J., SUMO modification of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis. Oncogene 2006, 25, (36), 4955-64. (41). Wang, H.; Ji, X.; Liu, X.; Yao, R.; Chi, J.; Liu, S.; Wang, Y.; Cao, W.; Zhou, Q.,

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Lentivirus-mediated inhibition of USP39 suppresses the growth of breast cancer cells in vitro. Oncol. Rep. 2013, 30, (6), 2871-7.

FIGURE LEGENDS Figure 1. SI2 inhibits the activity of SENPs. (A) The chemical structure of SI2. (B) The effect of 50 µM SI2 and SI30 on the isopeptidase activity of SENP1C was determined by in vitro gel-based SENP activity assay as described in EXPERIMENTAL PROCEDURES. (C) In an in vitro gel-based SENP activity assay, various concentrations of SI2 were pre-incubated with 5 nM SENP1C before SUMO2-∆RanGAP1 was added. After incubation, the reactions were stopped and the products were separated by 12% SDS-PAGE and visualized by coomassie brilliant blue (G250). The bands were scanned for densitometric analysis and the dose-response curve was shown. (D) Binding mode of SI2 in SENP1 catalytic site from docking study. SENP1C and SUMO1 were shown in gray and cyan, respectively. W465, F496, H529, and C603 were shown in stick form with carbon atoms colored in gray. SI2 was shown in stick form with carbon atoms colored in yellow. The SENP1 residues in 5 Å of SUMO1 residues 94-98 were shown as surface. The hydrogen bond was indicated by dashed lines. (E-G) The effect of 50 µM or 100 µM SI2 on the activity of cathepsin B (E) , cathepsin D (F) or proteasome (G) was determined as described in EXPERIMENTAL PROCEDURES. 50 µM E64 (inhibitor of Cathepsin B), 50 µM Pepstatin A (inhibitor of Cathepsin D), and 10 µM Epoxomicin (inhibitor of proteasome) were used as positive control, respectively. (H) HEK293T cells were

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transiently transfected with Flag-SUMO2 and empty vector or SENP1 for 24 h and then treated with DMSO or 5, 10, 20 µM SI2 for 2 h, and the indicated proteins were detected by western blotting. Figure 2. SI2 increases the SUMO-conjugated protein level in PC3 cells. PC3 cells stably expressing Flag-tagged SUMO1 (A), SUMO2 (B), and SUMO3 (C) were treated with DMSO and 10 µM, 20 µM SI2 for 1 h (A) or 2 h (B and C). The indicated proteins were detected by western blotting with anti-Flag antibody. Figure 3. Quantitative proteomic strategy to identify SUMOylated proteins. (A) In the schematic of experimental workflow, 12C14N labeled PC3-Flag-SUMO cells were treated with DMSO, and 13C15N labeled PC3-Flag-SUMO cells were treated with 20 µM SI2 for 1 h (SUMO1) or 2 h (SUMO2 and SUMO3). Equal amounts of lysates from DMSO (L) and SI2 (H) treatment were mixed. Proteins conjugated to Flag-SUMO were affinity-purified by anti-Flag antibody M2 beads. The M2 beads-enriched SUMO-targeted proteins were separated by 8% SDS-PAGE and stained by coomassie brilliant blue. The gel lanes were cut and the proteins were identified by mass spectrometry. (B-D) Extracts from DMSO-treated (DMSOL) and SI2-treated (SI2H) cells and M2 beads-enriched SUMO-targeted proteins from equal amounts of DMSOL, SI2H extracts (L&H IP) were separated by 8% SDS-PAGE and detected by anti-Flag antibody for Flag-SUMO1 (B), Flag-SUMO2 (C) and Flag-SUMO3 (D). (E-G) As described in (A), M2 beads-enriched SUMO1 (E) or SUMO2 (F) or SUMO3 (G) targeted proteins were separated by 8% SDS-PAGE and stained by coomassie brilliant blue. The gel lanes were cut in slices and the proteins

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were in-gel digested with trypsin and subjected to mass spectrometry analysis. Figure 4. Confirmation of target proteins of SUMO. The purified Flag-tagged USP39, HSPA1A or HSPA2 were incubated with SAE1/SAE2 fusion protein, His-UBC9, His-SUMO1/2/3 and ATP in the SUMOylation assay buffer. The reaction mixture was analyzed by western blotting with anti-His for SUMO1/2/3, UBC9 and anti-Flag for USP39 (A-C), HSPA1A (D) and HSPA2 (E). Figure 5. RS-like domain of USP39 contains its SUMOylation acceptor sites. (A) Structural diagram of USP39 and its truncations. (B) Western blotting analysis of the purified full-length USP39 protein and its truncations. (C) Purified full-length USP39 and its truncations were incubated with SAE1/SAE2, SUMO1 and UBC9 for SUMOylation assay and detected by western blotting. ∗ represents non-specific band. Figure 6. K6, K16, K29, K51 and K73 are the SUMO acceptor sites of USP39. (A) Six lysine residues in the RS-like domain of USP39 are shown. (B-C) Six mutants of full-length USP39 and the full length wild type USP39 (USP39-RSWT) were transfected into HEK293T cells. In each mutant, 5 of 6 lysines in the RS-like domain were mutated into arginines and the remaining lysine was indicated. Proteins were purified and subjected to in vitro SUMOylation assay. (D) Full-length USP39 (Flag-USP39WT) and USP39 with SUMO modification sites mutated (Flag-USP39SM) were transfected into HEK293T cells with vector or GFP-SUMO1, respectively. Cells were collected 48 h later. Flag-tagged proteins were purified by immunoprecipitation and were detected by western blotting using anti-Flag and anti-SUMO1 antibodies. * indicates SUMOylated USP39.

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Figure 7. SUMO modification sites mutated USP39 promotes prostate cancer cells proliferation. Empty vector, Flag-USP39WT and Flag-USP39SM were stably transfected into the hormone-independent prostate cancer cell line, PC3 (A) and hormone-dependent prostate cancer cell line, LNcap (C). Cell proliferation of PC3 (B) and LNcap (D) were monitored by CCK8 assay. The OD values on days 1, 2, 4, and 6 were normalized by dividing by the OD values at 10 h. All values represent means ± S.D. of three independent experiments, each performed in triplicate. ∗ indicates significant change (P < 0.05).

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Table I KEGG pathway analysis of SUMO substrates compared with genome background

KEGG pathway

KEGG pathway ID

p value

Gene

Spliceosome

hsa03040

1.26E-10

HSPA1A, HSPA1B, DDX5, XAB2, HNRNPU, HNRNPA3, EIF4A3, HNRNPM, SF3B1, HNRNPK, HSPA2, PCBP1, SNRNP200, USP39, DHX15, HNRNPC, ACIN1, PUF60, HSPA8, PRPF40A

Proteasome

hsa03050

6.20E-06

PSMD13, PSMC5, PSMD12, PSMD11, PSMC3, PSMA4, PSMA3, PSMD2, PSMD7

Glycolysis / Gluconeogenesis

hsa00010

2.91E-04

ALDOA, LDHA, PKM2, HK1, PDHA1, PGK1, GAPDH, ENO1

Valine, leucine and isoleucine biosynthesis

hsa00290

0.00147

IARS, LARS, PDHA1, VARS

Aminoacyl-tRNA biosynthesis

hsa00970

0.001814

IARS, TARS, LARS, FARSB, QARS, VARS

Pyruvate metabolism

hsa00620

0.010738

LDHA, PKM2, PDHA1, ACAT2, ACAT1

Valine, leucine and isoleucine degradation

hsa00280

0.014926

IVD, ACAT2, ACAT1, HADHA, HADHB

Propanoate metabolism

hsa00640

0.031725

LDHA, ACAT2, ACAT1, HADHA



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Figure 1. SI2 inhibits the activity of SENPs. (A) The chemical structure of SI2. (B) The effect of 50 µM SI2 and SI30 on the isopeptidase activity of SENP1C was determined by in vitro gel-based SENP activity assay as described in EXPERIMENTAL PROCEDURES. (C) In an in vitro gel-based SENP activity assay, various concentrations of SI2 were pre-incubated with 5 nM SENP1C before SUMO2-△RanGAP1 was added. After incubation, the reactions were stopped and the products were separated by 12% SDS-PAGE and visualized by coomassie brilliant blue (G250). The bands were scanned for densitometric analysis and the doseresponse curve was shown. (D) Binding mode of SI2 in SENP1 catalytic site from docking study. SENP1C and SUMO1 were shown in gray and cyan, respectively. W465, F496, H529, and C603 were shown in stick form with carbon atoms colored in gray. SI2 was shown in stick form with carbon atoms colored in yellow. The SENP1 residues in 5 Å of SUMO1 residues 94-98 were shown as surface. The hydrogen bond was indicated by dashed lines. (E-G) The effect of 50 µM or 100 µM SI2 on the activity of cathepsin B (E) , cathepsin D (F) or proteasome (G) was determined as described in EXPERIMENTAL PROCEDURES. 50 µM E64 (inhibitor of Cathepsin B), 50 µM Pepstatin A (inhibitor of Cathepsin D), and 10 µM Epoxomicin (inhibitor of proteasome) were used as positive control, respectively. (H)) HEK293T cells were transiently transfected with Flag-SUMO2 and empty vector or SENP1 for 24 h and then treated with DMSO or 5, 10, 20 µM SI2 for 2 h, and the indicated proteins were detected by western blotting. 160x99mm (300 x 300 DPI)


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Figure 2. SI2 increases the SUMO-conjugated protein level in PC3 cells. PC3 cells stably expressing Flagtagged SUMO1 (A), SUMO2 (B), and SUMO3 (C) were treated with DMSO and 10 µM, 20 µM SI2 for 1 h (A) or 2 h (B and C). The indicated proteins were detected by western blotting with anti-Flag antibody. 55x39mm (600 x 600 DPI)



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Figure 3. Quantitative proteomic strategy to identify SUMOylated proteins. (A) In the schematic of experimental workflow, 12C14N labeled PC3-Flag-SUMO cells were treated with DMSO, and 13C15N labeled PC3-Flag-SUMO cells were treated with 20 µM SI2 for 1 h (SUMO1), or 2 h (SUMO2) and (SUMO3). Equal amounts of lysates from DMSO (L) and SI2 (H) treatment were mixed. Proteins conjugated to Flag-SUMO were affinity-purified by anti-Flag antibody M2 beads. The M2 beads-enriched SUMO-targeted proteins were separated by 8%SDS-PAGE and stained by coomassie brilliant blue. The gel lanes were cut and the proteins were identified by mass spectrometry. (B-D) Extracts from DMSO-treated (DMSOL) and SI2-treated (SI2H) cells and M2 beads-enriched SUMO-targeted proteins from equal amounts of DMSOL, SI2H extracts (L&H IP) were separated by 8% SDS-PAGE and detected by anti-Flag antibody for Flag-SUMO1 (B), Flag-SUMO2 (C) and Flag-SUMO3 (D). (E-G) As described in (A), M2 beads-enriched SUMO1 (E) or SUMO2 (F) or SUMO3 (G) targeted proteins were separated by 8% SDS-PAGE and stained by coomassie brilliant blue. The gel lanes were cut in slices and the proteins were in-gel digested with trypsin and subjected to mass spectrometry analysis. 160x136mm (300 x 300 DPI)


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Figure 4. Confirmation of target proteins of SUMO. The purified Flag-tagged USP39, HSPA1A or HSPA2 were incubated with SAE1/SAE2 fusion protein, His-UBC9, His-SUMO1/2/3 and ATP in the SUMOylation assay buffer. The reaction mixture was analyzed by western blotting with anti-His for SUMO1/2/3, UBC9 and antiFlag for USP39 (A-C), HSPA1A (D) and HSPA2 (E). 56x40mm (600 x 600 DPI)



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Figure 5.RS-like domain of USP39 contains its SUMOylation acceptor sites. (A) Structural diagram of USP39 and its truncations. (B) Western blotting analysis of the purified full-length USP39 protein and its truncations. (C) Purified full-length USP39 and its truncations were incubated with SAE1/SAE2, SUMO1 and UBC9 for SUMOylation assay and detected by western blotting. * represents non-specific band. 36x17mm (600 x 600 DPI)


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Figure 6. K6, K16, K29, K51 and K73 are the SUMO acceptor sites of USP39. (A) Six lysine residues in the RS-like domain of USP39 are shown. (B-C) Six mutants of full-length USP39 and the full length wild type USP39 (USP39-RSWT) were transfected into HEK293T cells. In each mutant, 5 of 6 lysines in the RS-like domain were mutated into arginines and the remaining lysine was indicated. Proteins were purified and subjected to in vitro SUMOylation assay. (D) Full-length USP39 (Flag-USP39WT) and USP39 with SUMO modification sites mutated (Flag-USP39SM) were transfected into HEK293T cells with vector or GFP-SUMO1, respectively. Cells were collected 48 h later. Flag-tagged proteins were purified by immunoprecipitation and were detected by western blotting using anti-Flag and anti-SUMO1 antibodies. * indicates SUMOylated USP39. 57x42mm (600 x 600 DPI)



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Figure 7. SUMO modification sites mutated USP39 promotes prostate cancer cells proliferation. Empty vector, Flag-USP39WT and Flag-USP39SM were stably transfected into the hormone-independent prostate cancer cell line, PC3 (A) and hormone-dependent prostate cancer cell line, LNcap (C). Cell proliferation of PC3 (B) and LNcap (D) were monitored by CCK8 assay. The OD values on days 1, 2, 4, and 6 were normalized by dividing by the OD values at 10 h. All values represent means ± S.D. of three independent experiments, each performed in triplicate. * indicates significant change (P < 0.05). 60x45mm (600 x 600 DPI)


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49x51mm (600 x 600 DPI)


Important Role of SUMOylation of Spliceosome Factors in Prostate

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