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Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation
Biochemical and Biophysical Research Communications 470 (2016) 936e940
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation Jing Zheng a, b, Chunyan Tan a, b, Pengcheng Xue a, b, Jiakun Cao c, Feng Liu a, b, Ying Tan a, b, *, Yuyang Jiang b, d a
Department of Chemistry, Tsinghua University, Beijing 100084, China The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China c Shenzhen Kivita Innovative Drug Discovery Institute, Shenzhen 518055, Guangdong, China d Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2016 Accepted 25 January 2016 Available online 28 January 2016
Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. © 2016 Elsevier Inc. All rights reserved.
Keywords: Ubiquitination Proteolysis Proteasome PROTAP Bcl-xL
1. Introduction The intracellular protein levels are regulated through two major protein degradation pathways including ubiquitination proteasome pathway and autophagic degradation [1]. The ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo [2,3]. The selective degradation of specific proteins is extremely essential for many biological processes, especially the occurrence and development of diverse diseases [4e6]. UPP involves a cascade of enzymatic reactions catalyzed by the E1 ubiquitin activating enzyme, E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin-protein ligase [7,8]. The specificity of target protein is depending on the E3 ubiquitin ligases which transit polyubiquitin chain directly to the substrate [9]. Different from gene knockout, antisense, ribozyme, or RNAmediated interference (RNAi) technologies, degradation of specific protein through harnessing the ubiquitination technique
* Corresponding author. The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China. Tel.: þ86 0755 26036533. E-mail address: [email protected] (Y. Tan). http://dx.doi.org/10.1016/j.bbrc.2016.01.158 0006-291X/© 2016 Elsevier Inc. All rights reserved.
machinery attracts many scientists' attention. By simulating the natural process of UPP, some artificial systems were reported to recruit target proteins to E3 ubiquitin ligases to induce the polyubiquitination and protein degradation [10e13]. Zhou's group reported that fused proteins including two units: the target binding peptide (HPV-16 E7N) and E3 ligase (Cdc4pF/WD) could induce UPP and degrade target pRB protein [14]. Crews's group has used chimeric compounds, firstly named as Protein Targeting Chimeric molecules (PROTACS), to link a protein (MetAP-2) to the SCF complex for ubiquitination and degradation [12,13]. Yukihiro et al. induced ubiquitination-mediated degradation of cellular retinoic acid-binding proteins (CRABP-I and eII) using methyl bestatinligand hybrid molecules [15]. Although these strategies are proved to be effective for elimination of target proteins, they have some disadvantages. Firstly, molecular inhibitor binding with the active site of the protein may affect the protein structure and influence the recognition of E3 ligases. Secondly, few protein structure leads to design and synthesis of effective molecule inhibitors very hard. Thirdly, the synthesis difficulties in linking of molecular inhibitor and E3 recognition obstruct the development of PROTACS. By taking advantage of the strong yet selective interaction between peptide and protein [16], we designed a novel and general strategy, named Proteolysis Targeting Peptide (PROTAP), to
J. Zheng et al. / Biochemical and Biophysical Research Communications 470 (2016) 936e940
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selectively degrade the target proteins through UPP in this study. Unlike molecular inhibitor, peptide ligand will keep protein's original conformation and the interaction between protein and peptide has been studied deeply. So it is easier for us to get the peptide ligand to target protein that we are interested in. The new PROTAP strategy, composed of a target protein binding peptide, a linker and an ubiquitin E3 ligase recognition peptide. PROTAP strategy is proposed to recruit both target protein and E3 ligase, which in turn promotes the attachment of Ub to the target protein and leads to its degradation by proteosome. As a proof of the concept, Bcl-xL and VHL proteins are used as the model target protein and E3 ligase in this communication.
glycerol) added lysozyme (0.2 mg/ml) and PMSF (0.5 mM). Cellular debris was sonicated for 30 min was removed by centrifugation at 12,000 g for 30 min. The supernatant was loaded directly onto a Ni2þ-NTA column that was pre-equilibrated in lysis buffer and left at 4 C for 2 h. The Ni2þ-NTA column was then washed with wash buffer (50 mM Tris-Cl, pH ¼ 7.0, 100 mM NaCl, 25 mM imidazole and 10% glycerol). Proteins were eluted with elution buffer (50 mM Tris-Cl, pH ¼ 7.0, 100 mM NaCl, 200 mM imidazole and 10% glycerol). The protein concentration was determined using the Bradford method with bovine serum albumin (BSA) as a standard.
2. Materials and methods
The binding assay between AR-29 and Bcl-xL carried out by fluorescence polarization spectroscopy [19]. PROTAP AR-29 was labeled by fluorescein isothiocyanate (FITC), which has the maximum emission wavelength at 535 nm when excited at 485 nm. Increasing concentrations (0, 0.1, 1, 10, 50, 100, 500, 1000 and 2000 nM) of Bcl-xL protein was incubated with 12 nM FITC-AR-29 in PBS (pH ¼ 7.4) at 37 C for 30 min. The experimental data were fitted to the following equation,
2.1. Materials Anti-Bcl-xL polyclonal antibody (BS6453) was purchased from Bioworld Technology, Inc. The anti-Ubiquitin antibody [Ubi-1] (ab7254) was come from Abcam. Ubiquitin activating enzyme E1, ubiquitin conjugating enzyme UbcH5a (human), ubiquitin ligase VHL, 10 Ubiquitinylation buffer, Ubiquitin aldehyde, Mg2þ/ATP, HeLa S100 fraction and Proteasome 26S (human) were purchased from ENZO life science. Pyrophosphatase Inorganic (IPP) was provided by Life technologies. Other necessities were purchased from Sangon, Beyotime Biotechnology and so on. The proteolysis targeting peptide AR-29, FITC-AR-29 and RR-37 were synthesized by GL Biochem (Shanghai) Ltd.
2.5. Binding assay between AR-29 and Bcl-xL
P ¼ (I// I⊥)/(I// þ I⊥) where I//is the fluorescence intensity parallel to the excitation plane, I⊥ is the fluorescence intensity perpendicular to the excitation plane. 2.6. In vitro ubiquitination assay
2.2. Plasmid preparation DNA encoding for Bcl-xL was generated by PCR with HepG2 cell cDNA as template. The purified PCR product was digested by EcoR I/ Sac I mixture, and then ligated into the multiple cloning site of pET28a vector and pcDNA3.1 vector. The two recombinant plasmid pET-28a-Bcl-xL and pcDNA3.1-Bcl-xL were confirmed by automated direct sequencing. 2.3. Cell culture HCT116 and HEK293 cells were respectively grown in IMDM and DMEM containing 10% heat-inactivated fetal bovine serum at 37 C in a 5% CO2 atmosphere. Total proteins of HCT116 cells were isolated from the cells using lysis buffer (10 mM Hepes PH ¼ 7.9, 10 mM KCl, 1 mM EDTA, 0.1% NP-40, 1 mM PMSF, 1 mM Pepstatin, 1 mM Leupeptin, 0.5 mM Na3VO4 and ddH2O). Cell suspensions were sonicated for 3 s and suspended for 10 s for cycles. Then, they were put on ice for 30 min, inverting the tube every 5 min. The cell lysate was centrifuged at 20,000 g, 4 C for 15 min. Supernatant was collected for use. For transient transfection, HEK293 cells were seeded in 6well plate with 8.0 105 cell numbers and transfected with the pcDNA3.1-Bcl-xL using lipofectamine 2000 transfection reagent (Invitrogen). 2.4. Protein expression and purification The Bcl-xL was expressed and purified according to the previous reports [17,18]. The constructed plasmid was transformed into the BL21 (DE3) strain of E. coli. Cells transformed pET-28a-Bcl-xL were inoculated into 200 mL 2 YT grown with shaking at 37 C for hours until A600 ¼ 0.6. Then protein expression was induced by adding 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside) for another 4 h at 25 C. Induced cells were harvested by centrifugation at 5000 g for 15 min, and the cell pellet was then resuspended in lysis buffer (20 mM TriseHCl, pH ¼ 8.0, 500 mM NaCl and 10%
Firstly, we studied the time- and dose effect of AR-29. AR-29 was tested in the manual ubiquitination system containing 112 nM E1 (ubiquitin activating enzyme), 2.5 mM E2 (ubiquitin conjugating enzyme, UbcH5a), 0.25 mM E3 ligase (VHL), 5 mM Mg2þ/ATP, 0.4 U IPP, 1 mM DTT, 2.5 mM ubiquitin, 1 mg Bcl-xL and 1 ubiquitination buffer. Further study on the effect of AR-29 in a cellular ubquitination system was carried out. S100 extract separated from HeLa cell lysate and HCT116 lysate were chosen to verify the induced ubiquitin capacity of AR-29, including E1, E2, E3, and 26S proteasome. Then, the target protein Bcl-xL, ATP, PROTAP AR-29 and cell lysate were mixed. These results were tested by western blot. 2.7. Ubiquitination assay in cells To test whether the PROTAP strategy would work as effectively in cells as in vitro, we added eight D-arginine residues in the Nterminal of AR-29 to enable the peptide to permeate cell membrane and the peptide was named RR-37. pcDNA3.1-Bcl-xL was transfected into HEK293 cells by transient transfection. After 24 h incubation, cells were treated with increasing concentration (0, 10, 100, 200, 300 mM) of RR-37 for 8 h. 2.8. Western blot After ubiquitination assay in vitro or in cellular, proteins were separated by 12% SDS-PAGD and then transferred onto a polyvinlidene fluoride membrane (PVDF, 0.22 mm). Membranes were blocked using 0.5% skim milk for 1.5 h, and incubated with indicated primary antibodies including anti-Bcl-xL polyclonal antibody (1:1000) and anti-Ubiquitin antibody (1:1000) overnight at 4 C. After 3 times washes, the membranes were incubated with HRPlabeled secondary antibody (HRP, Horseradish Peroxidase) for 1.5 h. After another 3 times washes, the expression levels of proteins were detected using Gel imaging system and then were analyzed with Image J software.
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3. Results and discussion 3.1. AR-29 efficiently binding with Bcl-xL We designed new PROTAP strategy, composed of a target protein binding peptide, a linker and an ubiquitin E3 ligase recognition peptide (Fig. 1A). For proof of this concept, Bcl-xL was selected as a target protein. Bcl-xL is one of the important members of the Bcl-2 protein family, which is the key regulator of apoptosis [20]. In many kinds of cancer, the excessive Bcl-xL protein affects apoptosis pathway. Bcl-xL is also the most important characterization of certain cancer treatment responses and prognoses [20e22]. Binding peptides of Bcl-xL have been intensively studied and used in anti-apopotosis drug discovery, as well as in vitro screening of BclxL inhibitors [23,24]. The peptide (H-GQVGRQLAIIGDAINR-OH) was reported to be able to bind with Bcl-xL with the dissociation constant in a nM range [25,26]. A heptapeptide (ALAPYIP) was reported to be the ligand for the von Hippel-Lindau tumor suppressor protein (VHL) which is a part of the VBC-Cul2 E3 ubiquitin ligase complex [27,28] and was usually used in PROTACS study [10,12,15,29]. Therefore, the heptapeptide ALAPYIP was chosen as the VHL recognition domain in the current work [13,15,30,31]. For a general design, a hexapeptide (GGGGGG) was used as the linker to connect the two recognition domains [32]. The complete peptide was designated as “AR-29”, and the amino acid sequence of AR-29 is shown in Fig. 1B. The studies of PROTACS proved that the heptapeptide (ALAPYIP) linking with the target recognition domain binds efficiently with the VHL protein. In this work, the binding effect between AR-29 and the target Bcl-xL was identified by fluorescent polarization spectroscopy [33]. As shown in Fig. 1C, the value of fluorescence polarization changed with the increasing Bcl-xL concentration. The Kd value was determined to be about 58.7 nM, which is comparable to the literature report [26]. This result highly suggested that AR-29 could recognize and bind efficiently with the target protein Bcl-xL.
3.2. AR-29 induce Bcl-xL degradation through ubiquitination proteasome pathway in vitro Next, we tested the time- and dose-dependent effect of AR-29 in in vitro ubiquitination system. As shown in Fig. S1, AR-29 (from 1 mM to 250 mM) induced significant ubiquitination in a dosedependent manner. Then we chose 100 mM as the best concentration condition, the incubation time was studied from 6 h to 48 h.
The result revealed that the optimum incubation time was 24 h (Fig. S2). We performed western blot to prove the validity of the PROTAP strategy. As shown in Fig. 2, AR-29 induced the polyubiquitination of Bcl-xL which was detected by both Bcl-xL and ubiquitin antibodies. And the polyubiquitination Bcl-xL was degraded when added 26S proteasome. The in vitro experiment also proved that the PROTAP strategy has great potential for target protein degradation though the ubiquitination system. Further study was carried out to determine the effect of AR-29 in cell lysate system. The induced ubiquitination capacity of AR-29 was performed in S100 extract separated from HeLa cell including E1, E2, E3 and 26S proteasome [30]. The quantity of Bcl-xL protein decreased about 47.5% compared with the control when 100 mM AR-29 was added in the reaction system (Fig. 3A and B). Similar results were observed in human colon cancer HCT116 cell lysate. AR-29 also induced polyubiquitination of Bcl-xL, resulting in degradation by 26S proteasome (Fig. S3). Both the S100 extract and HCT116 cell lysate experiments suggested that our PROTAP strategy has great potential for in vivo system in further applications.
3.3. AR-29 induce Bcl-xL ubiquitination and degradation in cells To apply the PROTAP strategy in cells, we added a poly-D-arginine tag (mimic of HIV TAT sequence) in the N-terminal of AR-29 which permits cell permeability of the peptide [13,34]. The new peptide using in cells was named RR-37 (Fig. 4A). An increased expression of Bcl-xL was observed in HEK293 cells transfected pcDNA 3.1-Bcl-xL (Fig. 4B). At 24 h after transfection, we treated cells with indicated concentration of RR-37 for 8 h. The levels of BclxL protein decreased with increasing concentration of RR-37 (Fig. 4C). When applied 300 mM RR-37 to the cells, the protein level of Bcl-xL reduced more than 50% compared with the control (Fig. 4D). These results indicated that our novel PROTAP is a promising strategy for reducing target protein through UPP in vivo. In summary, we designed and synthesized AR-29, which consists of a Bcl-xL binding peptide, a peptide linker, and an E3 ligase recognition peptide unit, to successfully induce the ubiquitination and degradation of Bcl-xL protein in vitro and in cells. The PROTAP strategy harnessing UPP pathway was proved to be a simple and general method with high specificity to knock down proteins. By simply replacing the ligand of targets with corresponding peptides, this strategy might be generalized to other disease-related protein targets. And the strategy has great potential for degradation target
Fig. 1. (A) The principle of PROTAP inducing ubiquitination and degradation of target protein (B) Components and amino acid sequence of PROTAP AR-29. (C) Binding affinity of FITC labeled AR-29 with Bcl-xL by fluorescence polarization assay. The different concentrations of Bcl-xL: 0, 0.1, 1, 10, 50, 100, 500, 1000 and 2000 nM.
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Fig. 2. Western-blot results of ubiquitination and degradation of Bcl-xL induced by AR-29. (A) incubated with antibody to Bcl-xL and (B) incubated with antibody to ubiquitin indicated that AR-29 induced the ubquitination of Bcl-xL. (C) Ubiquitinated Bcl-xL can be identified and eliminated by 26S proteasome.
Fig. 3. (A) Western-blot of ubiquitination and degradation of Bcl-xL in S100 extract system induced by AR-29. (B) AR-29 induced obvious reduction of Bcl-xL, decreasing about 50%.
Fig. 4. The effect of RR-37 in HEK293 cells. (A) Components and amino acid sequence of PROTAP RR-37. (B) Expression of pcDNA3.1-Bcl-xL in HEK293 cells. (C) The decreasing levels of Bcl-xL protein in cells after treating with increasing concentration of RR-37 for 8 h (D) RR-37 induced obvious reduction of Bcl-xL, decreasing about 50%.
proteins in vivo. Conflict of interest
Foundation of China (No. 21302108, 21572115) and Shenzhen Municipal government (JSGG20141016150327538, CXB201104210014A).
The authors declare no conflict of interest. Appendix A. Supplementary data Acknowledgments This work is supported by grants from National Natural Science
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.01.158.
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J. Zheng et al. / Biochemical and Biophysical Research Communications 470 (2016) 936e940
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation Jing Zheng a, b, Chunyan Tan a, b, Pengcheng Xue a, b, Jiakun Cao c, Feng Liu a, b, Ying Tan a, b, *, Yuyang Jiang b, d a
Department of Chemistry, Tsinghua University, Beijing 100084, China The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China c Shenzhen Kivita Innovative Drug Discovery Institute, Shenzhen 518055, Guangdong, China d Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2016 Accepted 25 January 2016 Available online 28 January 2016
Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. © 2016 Elsevier Inc. All rights reserved.
Keywords: Ubiquitination Proteolysis Proteasome PROTAP Bcl-xL
1. Introduction The intracellular protein levels are regulated through two major protein degradation pathways including ubiquitination proteasome pathway and autophagic degradation [1]. The ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo [2,3]. The selective degradation of specific proteins is extremely essential for many biological processes, especially the occurrence and development of diverse diseases [4e6]. UPP involves a cascade of enzymatic reactions catalyzed by the E1 ubiquitin activating enzyme, E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin-protein ligase [7,8]. The specificity of target protein is depending on the E3 ubiquitin ligases which transit polyubiquitin chain directly to the substrate [9]. Different from gene knockout, antisense, ribozyme, or RNAmediated interference (RNAi) technologies, degradation of specific protein through harnessing the ubiquitination technique
* Corresponding author. The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China. Tel.: þ86 0755 26036533. E-mail address: [email protected] (Y. Tan). http://dx.doi.org/10.1016/j.bbrc.2016.01.158 0006-291X/© 2016 Elsevier Inc. All rights reserved.
machinery attracts many scientists' attention. By simulating the natural process of UPP, some artificial systems were reported to recruit target proteins to E3 ubiquitin ligases to induce the polyubiquitination and protein degradation [10e13]. Zhou's group reported that fused proteins including two units: the target binding peptide (HPV-16 E7N) and E3 ligase (Cdc4pF/WD) could induce UPP and degrade target pRB protein [14]. Crews's group has used chimeric compounds, firstly named as Protein Targeting Chimeric molecules (PROTACS), to link a protein (MetAP-2) to the SCF complex for ubiquitination and degradation [12,13]. Yukihiro et al. induced ubiquitination-mediated degradation of cellular retinoic acid-binding proteins (CRABP-I and eII) using methyl bestatinligand hybrid molecules [15]. Although these strategies are proved to be effective for elimination of target proteins, they have some disadvantages. Firstly, molecular inhibitor binding with the active site of the protein may affect the protein structure and influence the recognition of E3 ligases. Secondly, few protein structure leads to design and synthesis of effective molecule inhibitors very hard. Thirdly, the synthesis difficulties in linking of molecular inhibitor and E3 recognition obstruct the development of PROTACS. By taking advantage of the strong yet selective interaction between peptide and protein [16], we designed a novel and general strategy, named Proteolysis Targeting Peptide (PROTAP), to
J. Zheng et al. / Biochemical and Biophysical Research Communications 470 (2016) 936e940
937
selectively degrade the target proteins through UPP in this study. Unlike molecular inhibitor, peptide ligand will keep protein's original conformation and the interaction between protein and peptide has been studied deeply. So it is easier for us to get the peptide ligand to target protein that we are interested in. The new PROTAP strategy, composed of a target protein binding peptide, a linker and an ubiquitin E3 ligase recognition peptide. PROTAP strategy is proposed to recruit both target protein and E3 ligase, which in turn promotes the attachment of Ub to the target protein and leads to its degradation by proteosome. As a proof of the concept, Bcl-xL and VHL proteins are used as the model target protein and E3 ligase in this communication.
glycerol) added lysozyme (0.2 mg/ml) and PMSF (0.5 mM). Cellular debris was sonicated for 30 min was removed by centrifugation at 12,000 g for 30 min. The supernatant was loaded directly onto a Ni2þ-NTA column that was pre-equilibrated in lysis buffer and left at 4 C for 2 h. The Ni2þ-NTA column was then washed with wash buffer (50 mM Tris-Cl, pH ¼ 7.0, 100 mM NaCl, 25 mM imidazole and 10% glycerol). Proteins were eluted with elution buffer (50 mM Tris-Cl, pH ¼ 7.0, 100 mM NaCl, 200 mM imidazole and 10% glycerol). The protein concentration was determined using the Bradford method with bovine serum albumin (BSA) as a standard.
2. Materials and methods
The binding assay between AR-29 and Bcl-xL carried out by fluorescence polarization spectroscopy [19]. PROTAP AR-29 was labeled by fluorescein isothiocyanate (FITC), which has the maximum emission wavelength at 535 nm when excited at 485 nm. Increasing concentrations (0, 0.1, 1, 10, 50, 100, 500, 1000 and 2000 nM) of Bcl-xL protein was incubated with 12 nM FITC-AR-29 in PBS (pH ¼ 7.4) at 37 C for 30 min. The experimental data were fitted to the following equation,
2.1. Materials Anti-Bcl-xL polyclonal antibody (BS6453) was purchased from Bioworld Technology, Inc. The anti-Ubiquitin antibody [Ubi-1] (ab7254) was come from Abcam. Ubiquitin activating enzyme E1, ubiquitin conjugating enzyme UbcH5a (human), ubiquitin ligase VHL, 10 Ubiquitinylation buffer, Ubiquitin aldehyde, Mg2þ/ATP, HeLa S100 fraction and Proteasome 26S (human) were purchased from ENZO life science. Pyrophosphatase Inorganic (IPP) was provided by Life technologies. Other necessities were purchased from Sangon, Beyotime Biotechnology and so on. The proteolysis targeting peptide AR-29, FITC-AR-29 and RR-37 were synthesized by GL Biochem (Shanghai) Ltd.
2.5. Binding assay between AR-29 and Bcl-xL
P ¼ (I// I⊥)/(I// þ I⊥) where I//is the fluorescence intensity parallel to the excitation plane, I⊥ is the fluorescence intensity perpendicular to the excitation plane. 2.6. In vitro ubiquitination assay
2.2. Plasmid preparation DNA encoding for Bcl-xL was generated by PCR with HepG2 cell cDNA as template. The purified PCR product was digested by EcoR I/ Sac I mixture, and then ligated into the multiple cloning site of pET28a vector and pcDNA3.1 vector. The two recombinant plasmid pET-28a-Bcl-xL and pcDNA3.1-Bcl-xL were confirmed by automated direct sequencing. 2.3. Cell culture HCT116 and HEK293 cells were respectively grown in IMDM and DMEM containing 10% heat-inactivated fetal bovine serum at 37 C in a 5% CO2 atmosphere. Total proteins of HCT116 cells were isolated from the cells using lysis buffer (10 mM Hepes PH ¼ 7.9, 10 mM KCl, 1 mM EDTA, 0.1% NP-40, 1 mM PMSF, 1 mM Pepstatin, 1 mM Leupeptin, 0.5 mM Na3VO4 and ddH2O). Cell suspensions were sonicated for 3 s and suspended for 10 s for cycles. Then, they were put on ice for 30 min, inverting the tube every 5 min. The cell lysate was centrifuged at 20,000 g, 4 C for 15 min. Supernatant was collected for use. For transient transfection, HEK293 cells were seeded in 6well plate with 8.0 105 cell numbers and transfected with the pcDNA3.1-Bcl-xL using lipofectamine 2000 transfection reagent (Invitrogen). 2.4. Protein expression and purification The Bcl-xL was expressed and purified according to the previous reports [17,18]. The constructed plasmid was transformed into the BL21 (DE3) strain of E. coli. Cells transformed pET-28a-Bcl-xL were inoculated into 200 mL 2 YT grown with shaking at 37 C for hours until A600 ¼ 0.6. Then protein expression was induced by adding 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside) for another 4 h at 25 C. Induced cells were harvested by centrifugation at 5000 g for 15 min, and the cell pellet was then resuspended in lysis buffer (20 mM TriseHCl, pH ¼ 8.0, 500 mM NaCl and 10%
Firstly, we studied the time- and dose effect of AR-29. AR-29 was tested in the manual ubiquitination system containing 112 nM E1 (ubiquitin activating enzyme), 2.5 mM E2 (ubiquitin conjugating enzyme, UbcH5a), 0.25 mM E3 ligase (VHL), 5 mM Mg2þ/ATP, 0.4 U IPP, 1 mM DTT, 2.5 mM ubiquitin, 1 mg Bcl-xL and 1 ubiquitination buffer. Further study on the effect of AR-29 in a cellular ubquitination system was carried out. S100 extract separated from HeLa cell lysate and HCT116 lysate were chosen to verify the induced ubiquitin capacity of AR-29, including E1, E2, E3, and 26S proteasome. Then, the target protein Bcl-xL, ATP, PROTAP AR-29 and cell lysate were mixed. These results were tested by western blot. 2.7. Ubiquitination assay in cells To test whether the PROTAP strategy would work as effectively in cells as in vitro, we added eight D-arginine residues in the Nterminal of AR-29 to enable the peptide to permeate cell membrane and the peptide was named RR-37. pcDNA3.1-Bcl-xL was transfected into HEK293 cells by transient transfection. After 24 h incubation, cells were treated with increasing concentration (0, 10, 100, 200, 300 mM) of RR-37 for 8 h. 2.8. Western blot After ubiquitination assay in vitro or in cellular, proteins were separated by 12% SDS-PAGD and then transferred onto a polyvinlidene fluoride membrane (PVDF, 0.22 mm). Membranes were blocked using 0.5% skim milk for 1.5 h, and incubated with indicated primary antibodies including anti-Bcl-xL polyclonal antibody (1:1000) and anti-Ubiquitin antibody (1:1000) overnight at 4 C. After 3 times washes, the membranes were incubated with HRPlabeled secondary antibody (HRP, Horseradish Peroxidase) for 1.5 h. After another 3 times washes, the expression levels of proteins were detected using Gel imaging system and then were analyzed with Image J software.
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3. Results and discussion 3.1. AR-29 efficiently binding with Bcl-xL We designed new PROTAP strategy, composed of a target protein binding peptide, a linker and an ubiquitin E3 ligase recognition peptide (Fig. 1A). For proof of this concept, Bcl-xL was selected as a target protein. Bcl-xL is one of the important members of the Bcl-2 protein family, which is the key regulator of apoptosis [20]. In many kinds of cancer, the excessive Bcl-xL protein affects apoptosis pathway. Bcl-xL is also the most important characterization of certain cancer treatment responses and prognoses [20e22]. Binding peptides of Bcl-xL have been intensively studied and used in anti-apopotosis drug discovery, as well as in vitro screening of BclxL inhibitors [23,24]. The peptide (H-GQVGRQLAIIGDAINR-OH) was reported to be able to bind with Bcl-xL with the dissociation constant in a nM range [25,26]. A heptapeptide (ALAPYIP) was reported to be the ligand for the von Hippel-Lindau tumor suppressor protein (VHL) which is a part of the VBC-Cul2 E3 ubiquitin ligase complex [27,28] and was usually used in PROTACS study [10,12,15,29]. Therefore, the heptapeptide ALAPYIP was chosen as the VHL recognition domain in the current work [13,15,30,31]. For a general design, a hexapeptide (GGGGGG) was used as the linker to connect the two recognition domains [32]. The complete peptide was designated as “AR-29”, and the amino acid sequence of AR-29 is shown in Fig. 1B. The studies of PROTACS proved that the heptapeptide (ALAPYIP) linking with the target recognition domain binds efficiently with the VHL protein. In this work, the binding effect between AR-29 and the target Bcl-xL was identified by fluorescent polarization spectroscopy [33]. As shown in Fig. 1C, the value of fluorescence polarization changed with the increasing Bcl-xL concentration. The Kd value was determined to be about 58.7 nM, which is comparable to the literature report [26]. This result highly suggested that AR-29 could recognize and bind efficiently with the target protein Bcl-xL.
3.2. AR-29 induce Bcl-xL degradation through ubiquitination proteasome pathway in vitro Next, we tested the time- and dose-dependent effect of AR-29 in in vitro ubiquitination system. As shown in Fig. S1, AR-29 (from 1 mM to 250 mM) induced significant ubiquitination in a dosedependent manner. Then we chose 100 mM as the best concentration condition, the incubation time was studied from 6 h to 48 h.
The result revealed that the optimum incubation time was 24 h (Fig. S2). We performed western blot to prove the validity of the PROTAP strategy. As shown in Fig. 2, AR-29 induced the polyubiquitination of Bcl-xL which was detected by both Bcl-xL and ubiquitin antibodies. And the polyubiquitination Bcl-xL was degraded when added 26S proteasome. The in vitro experiment also proved that the PROTAP strategy has great potential for target protein degradation though the ubiquitination system. Further study was carried out to determine the effect of AR-29 in cell lysate system. The induced ubiquitination capacity of AR-29 was performed in S100 extract separated from HeLa cell including E1, E2, E3 and 26S proteasome [30]. The quantity of Bcl-xL protein decreased about 47.5% compared with the control when 100 mM AR-29 was added in the reaction system (Fig. 3A and B). Similar results were observed in human colon cancer HCT116 cell lysate. AR-29 also induced polyubiquitination of Bcl-xL, resulting in degradation by 26S proteasome (Fig. S3). Both the S100 extract and HCT116 cell lysate experiments suggested that our PROTAP strategy has great potential for in vivo system in further applications.
3.3. AR-29 induce Bcl-xL ubiquitination and degradation in cells To apply the PROTAP strategy in cells, we added a poly-D-arginine tag (mimic of HIV TAT sequence) in the N-terminal of AR-29 which permits cell permeability of the peptide [13,34]. The new peptide using in cells was named RR-37 (Fig. 4A). An increased expression of Bcl-xL was observed in HEK293 cells transfected pcDNA 3.1-Bcl-xL (Fig. 4B). At 24 h after transfection, we treated cells with indicated concentration of RR-37 for 8 h. The levels of BclxL protein decreased with increasing concentration of RR-37 (Fig. 4C). When applied 300 mM RR-37 to the cells, the protein level of Bcl-xL reduced more than 50% compared with the control (Fig. 4D). These results indicated that our novel PROTAP is a promising strategy for reducing target protein through UPP in vivo. In summary, we designed and synthesized AR-29, which consists of a Bcl-xL binding peptide, a peptide linker, and an E3 ligase recognition peptide unit, to successfully induce the ubiquitination and degradation of Bcl-xL protein in vitro and in cells. The PROTAP strategy harnessing UPP pathway was proved to be a simple and general method with high specificity to knock down proteins. By simply replacing the ligand of targets with corresponding peptides, this strategy might be generalized to other disease-related protein targets. And the strategy has great potential for degradation target
Fig. 1. (A) The principle of PROTAP inducing ubiquitination and degradation of target protein (B) Components and amino acid sequence of PROTAP AR-29. (C) Binding affinity of FITC labeled AR-29 with Bcl-xL by fluorescence polarization assay. The different concentrations of Bcl-xL: 0, 0.1, 1, 10, 50, 100, 500, 1000 and 2000 nM.
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Fig. 2. Western-blot results of ubiquitination and degradation of Bcl-xL induced by AR-29. (A) incubated with antibody to Bcl-xL and (B) incubated with antibody to ubiquitin indicated that AR-29 induced the ubquitination of Bcl-xL. (C) Ubiquitinated Bcl-xL can be identified and eliminated by 26S proteasome.
Fig. 3. (A) Western-blot of ubiquitination and degradation of Bcl-xL in S100 extract system induced by AR-29. (B) AR-29 induced obvious reduction of Bcl-xL, decreasing about 50%.
Fig. 4. The effect of RR-37 in HEK293 cells. (A) Components and amino acid sequence of PROTAP RR-37. (B) Expression of pcDNA3.1-Bcl-xL in HEK293 cells. (C) The decreasing levels of Bcl-xL protein in cells after treating with increasing concentration of RR-37 for 8 h (D) RR-37 induced obvious reduction of Bcl-xL, decreasing about 50%.
proteins in vivo. Conflict of interest
Foundation of China (No. 21302108, 21572115) and Shenzhen Municipal government (JSGG20141016150327538, CXB201104210014A).
The authors declare no conflict of interest. Appendix A. Supplementary data Acknowledgments This work is supported by grants from National Natural Science
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.01.158.
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