Cellular Signalling 26 (2014) 2266–2275
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The catalytic triad of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation Zhen Bo Song a,b, Biao Liu a,c,d, Yu Yin Li a,b, Ping Wu b, Yong Li Bao a,⁎, Yan Xin Huang b,c, Yin Wu b, Lu Guo Sun a,b, Chun Lei Yu a,b, Ying Sun c, Li Hua Zheng c, Yu Xin Li b,c,⁎ a
National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China d Department of Handsurgery, China–Japan Union Hospital, Jilin University, Changchun 130033, China b c
a r t i c l e
i n f o
Article history: Received 14 May 2014 Received in revised form 22 June 2014 Accepted 4 July 2014 Available online 15 July 2014 Keywords: TSP50 Serine protease Catalytic triad Mutants Cell proliferation Mechanism
a b s t r a c t Testes-specific protease 50 (TSP50) is a novelly identified pro-oncogene and it shares a similar enzymatic structure with many serine proteases. Our previous results suggested that TSP50 could promote tumorigenesis through degradation of IκBα protein and activating NF-κB signaling, and the threonine mutation in its catalytic triad could depress TSP50-mediated cell proliferation. However, whether the two other residues in the catalytic triad of TSP50 play a role in maintaining protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. Here, we constructed and characterized three catalytic triad mutants of TSP50 and found that all the mutants could significantly depress TSP50-induced cell proliferation and colony formation in vitro and tumor formation in vivo, and the aspartic acid at position 206 in the catalytic triad played a more crucial role than threonine and histidine in this process. Mechanistic studies revealed that the mutants in the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. Furthermore, our data indicated that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Most importantly, we demonstrated that TSP50 could interact with IκBα protein and cleave it directly as a new protease in vitro. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Serine proteases contain a large family of enzymes that are characterized by possessing an active serine in their catalytic site, and they play a critical role in numerous physiologic processes including digestion, blood coagulation, complement activation, fibrinolysis, reproduction, embryonic development, protein processing and tissue remodeling [1–6]. Serine proteases maintain a strictly conserved active site geometry among their catalytic Ser, His and Asp residues; this
Abbreviations: Bcl-2, B cell lymphoma/leukemia-2; BrdU, 5-bromo-2′-deoxyuridine; CHO cell, Chinese-hamster ovary cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK 293T, human embryonic kidney 293T cells; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear factor κB; PMSF, phenylmethanesulphonyl fluoride; PMA, phorbol myristate acetate; RT-PCR, reverse transcription-PCR;TSP50, testes-specific protease 50. ⁎ Corresponding authors. Tel.: +86 431 8509 8455; fax: +86 431 8916 5917. E-mail addresses: [email protected] (Z.B. Song), [email protected] (B. Liu), [email protected] (Y.Y. Li), [email protected] (P. Wu), [email protected] (Y.L. Bao), [email protected] (Y.X. Huang), [email protected] (Y. Wu), [email protected] (L.G. Sun), [email protected] (C.L. Yu), [email protected] (Y. Sun), [email protected] (L.H. Zheng), [email protected] (Y.X. Li).
http://dx.doi.org/10.1016/j.cellsig.2014.07.012 0898-6568/© 2014 Elsevier Inc. All rights reserved.
shared catalytic structure suggests that common architectural motifs are likely to be found in the molecular designs of active sites utilizing a Ser-His-Asp triad [5]. The substitution of either His57 or Ser195 to Ala is sufficient to completely disable the catalytic triad, and the substitution of Asp102 to Asn in trypsin decreases kcat/Km by 104 at neutral pH [7]. Increasing evidences indicates that some serine proteases, which have been traditionally viewed as degradative enzymes, are also signaling molecules that regulate multiple cellular functions by activating specific receptors [8–10], protease receptors such as PAR-1, PAR-3 and PAR-2, were cleaved and activated by thrombin and trypsin-like enzymes respectively [11–13]. TSP50 is a testis-specific gene encoding a protein that is homologous to serine proteases, and it shares two critical catalytic triads, histidine and aspartic acid at positions 153 and 206, respectively. However, the most crucial triad, serine, at position 310 was replaced by threonine and it possessed enzymatic activity [14,15], suggesting that TSP50 might represent a novel type of protease. When the catalytic threonine residual of TSP50 is replaced by alanine, it was unable to cleave β-casein [15]. Our recent results showed that TSP50 T310A mutation significantly depressed TSP50-induced cell proliferation, colony formation, resisting apoptosis, cell migration and cell invasion in vitro and abolished the oncogenicity of TSP50 in vivo [16]. These results strongly indicated
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that the threonine in the catalytic triad of TSP50 is essential to its function in tumorigenesis. However, whether this is correlated to protease activity of TSP50, the role of the two other residues in catalytic triad of TSP50 in protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. In addition to expression in normal testes, TSP50 was abnormally expressed at high level in many breast cancer and colorectal carcinoma biopsies tested [17–19]. Previously, we revealed that overexpression of TSP50 efficiently promotes cell proliferation and tumor formation through the activation of the NF-κB signaling pathway, and TSP50 can promote the degradation of κBα proteins by binding to the NF-κB: IκBα complex [20]. However, whether TSP50 can interact with IκBα directly and cleave it as a protease is unknown. In this paper, we mainly studied the effects of the histidine and aspartic acid mutation in the catalytic triad of TSP50 on its function in tumorigenesis and the mechanisms involved this process. We provided evidences that the mutations in the catalytic triad of TSP50 could significantly depressed TSP50-mediated cell proliferation and tumor formation, and aspartic acid at positions 206 played a much more important role in maintaining the function of TSP50.
using LipofectamineTM2000 (Invitrogen), according to the instructions of the manufacturer. Transfected cells were incubated in the presence of G418 for 2 weeks and the stable transfected cell lines were selected.
2. Materials and methods
Cytosolic and nuclear extracts were prepared and Western blotting was performed as described previously [22].
2.4. RNA extract and RT-PCR Total RNA was prepared from cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The RT-PCR kit was bought from transGgenbiotech. Total RNA (3 μg) was reverse transcribed into cDNA by incubating at 50 °C for 60 min. TSP50 mRNA was amplified from cDNA templates by RT-PCR using the primers 5′-CGGATCCATGCAGGGGAAGCC-3′ (sense) and 5′-GCTCTAGAAGTCAGAGGGCAG-3′ (antisense), β-actin primers 5′-TCGTGCGTGACATTAAGGAG-3′ (sense) and 5′-ATGCCAGGGTACAT GGTGGT-3′ (antisense). PCR was performed for 30 cycles (each cycle consisting of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s). The PCR products were analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide and visualized under UV light. 2.5. Western blotting assay
2.1. Antibodies and reagents 2.6. MTT assay Polyclonal antibodies against p65, IκBα, Histone1, C-Myc, COX-2, cyclinD1, Ki-67, β-actin and Bcl-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody against GAPDH was purchased from Kangcheng Biotech (Shanghai, China). Anti-TSP50 monoclonal antibody was prepared in our laboratory [21]. The site-directed Gene Mutagenesis Kit and the Calcium Phosphate Cell Transfection Kit were purchased from Beyotime (Shanghai, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The 5-bromo-2′-deoxyuridine (BrdU) labeling and detection ELISA kit was purchased from Roche Diagnostics (Mannheim, Germany). The Pierce Crosslink IP Kit was bought from Thermo Scientific (no: 26147).
Cell growth was determined by MTT assay. The cells were plated at 1 × 104 cells per well in 96-well microtiter plates. After incubation for the indicated time, MTT (5 mg/mL) was added to each well and incubated for 4 h. The absorbance was recorded on a microplate reader at a wavelength of 570 nm. 2.7. Colony formation in soft agar One milliliter base agar (1.2% agar plus 2 × DMEM in equal volumes) was added to six-well plates. Twenty four hours prior to initiating the soft agar assay, 4 × 103 cells/well were added in 1 mL of top agar (0.7% agar mixed with 2× DMEM in equal volumes) to each well. Plates were maintained at 37 °C for 14 days before colonies were counted.
2.2. Cell lines and cell culture 2.8. BrdU incorporation assay CHO cells (Chinese hamster ovary cells) and HEK 293T cells (human embryonic kidney 293T cells) were obtained from the Chinese Academy of Sciences Shanghai Institute for Biological Sciences-Cell Resource Center, which had characterized the cell lines by short tandem repeat profiling, cell morphology and karyotyping assay. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen, USA), which was supplemented with 10% fetal bovine serum (TBD Science, Tianjin, China), 100 U/mL penicillin and 100 μg/mL streptomycin (Ameresco, USA), at 37°C with 5% CO2.
DNA synthesis was assessed by measuring the incorporation of BrdU into newly synthesized strands. Cells were replated at 1 × 103 cells/well on 96-well plates. Twenty-four hours after plating, BrdU labeling was initiated by adding the labeling solution at a final concentration of 10 μM to the culture medium. After the cells were incubated for 6 h, labeling was stopped and BrdU uptake was measured according to the protocol of the manufacturer. 2.9. Analysis of luciferase activity
2.3. Plasmid constructs and transfection The EGFP fusion protein plasmids and the pNF-κB-luc plasmid were prepared in our laboratory. The three point mutation constructs of TSP50, T310A, D206A and H153A originated from the construct pcDNA3.0-TSP50 were achieved using Sit Directed Mutagenesis Kit. The primer sequences used were as follows: TSP50 T310A-For: 5′-GTTCTGCTATGAGCTAGCTGGAGAGCCCTTGGTC-3′, Rev: 5′-GACCAA GGGCTCTCCAGCTAGCTCATAGCAGAAC-3′, TSP50 D206A-For: 5′-GTGG GCCAGGCCAACGCCATCGGCCTCCTCAAG-3′, Rev: 5′-CTTGAGGAGGCC GATGGCGTTGGCCTGGCCCAC-3′, TSP50 H153A -For: 5′-GGTGCTGACT GTGGCCGCCTGCCTGATCTGGCGTG-3′, Rev: 5′-CACGCCAGATCAGGCA GGCGGCCACAGTCAGCACC-3′. The TSP50 and its point mutation constructs were cloned into the pcDNA3.0 basic vector. Cells were transfected with the T310A, D206A, and H153A plasmids per well
Firefly luciferase activity was measured 48 h after transfection. Analysis of luciferase activity was performed as described previously [23]. 2.10. Immunofluorescence detection The localization of p65 and Ki-67 staining were carried out in accordance with previously described procedures [24]. 2.11. Co-immunoprecipitation Co-immunoprecipitation experiments were performed by using the Pierce Crosslink Immunoprecipitation kit from Thermo Scientific according to the standard protocol of the manufacturer.
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2.12. Protein purification To purify endogenous TSP50 and its three mutants, TSP50 antibodyaffinity column was prepared by binding of antibody to protein A/G plus agarose and the bound antibody was crosslinked by the DSS crosslinker following the manufacturer's instructions. TSP50 was purified by passing the cell lysate through the TSP50 antibody-affinity column and eluting it with elution buffer provided in immunorecipitation kit.
2.13. Zymogram gel assay To test enzymatic activity, equal amounts of TSP50 and mutant TSP50 proteins were applied in duplicate to 12% Tris–glycine gels containing 1 mg/mL β-casein. After electrophoresis, the gels were washed with 2.5% Triton X-100 twice for 30 min, rinsed three times for 30 min with a 50 mM Tris–HCl buffer (pH 7.6) containing 5 mM CaCl2, 0.02% Brij-35, and 0.2% sodium azide, and then incubated overnight at 37 °C. After the enzymatic reactions, the gels were stained with Coomassie blue R-250 and destained with acetic acid and methanol. Areas of enzyme activity were detected as clear bands against the blue-stained background. To normalize the enzyme digested band created by TSP50, duplicate cell extracts were prepared for Western analysis. The experiments were done three times.
2.14. Tumorigenicity studies and immunohistochemistry assay Male BALB/c nude mice (6-weeks old) were purchased from the Chinese Academy of Sciences and were housed under pathogenfree conditions. Analysis of tumorigenicity and immunohistochemistry were performed as described previously [16]. Animal experiments were performed in accordance with established guidelines. The Chinese Academy of Sciences Animal Care and Use Committee gave approval for the animal experiments.
3.2. Effects of point mutations in catalytic triad on TSP50-mediated promotion of cell growth Previously, our results have shown that cell proliferation and colony formation were significantly promoted by overexpression of TSP50 in vitro [20,25], and TSP50 T310A mutation severely restrained cell proliferation [16]. Here, we mainly evaluated the effects of two other residues (Histidine153 and Aspartate206) in catalytic triad on TSP50mediated cell proliferation, and compared the function differences among the different TSP50 mutants (TSP50 T310A mutation is a positive control here). Results from the MTT cell proliferation assay demonstrated that expression of TSP50 T310A, TSP50 D206A and TSP50 H153A greatly destroyed the cell-proliferation-promoting activity by expression of TSP50. What is more, TSP50 D206A-expressing cells grew most slowly (Fig. 2A). Similarly, when DNA synthesis was measured by BrdU incorporation, we found that the three TSP50 mutants greatly decreased the uptake of BrdU in different degrees (Fig. 2B). Moreover, the anchorage-independent growth ability of TSP50 and the mutantexpressing cells was assayed by colony formation in soft agar, where the number of colonies obtained in the TSP50 mutant-expressing cultures was significantly lower than the number obtained in the TSP50-expressing cells; furthermore, TSP50 D206A-expressing cells formed much less colonies than those by two other mutants (Fig. 2C and D). Antigen Ki-67 is a nuclear protein and it is strictly associated with cell proliferation. Fluorescence assay suggested that the three TSP50 mutants strongly impaired the expression of Ki-67 induced by TSP50 (Fig. 2E and F). Overall, our results suggested that all three residues in the catalytic triad were indispensable for TSP50-mediated cell growth, and aspartic acid at position 206 may play a crucial role. 3.3. Abolishment of TSP50-mediated tumorigenicity by point mutations in its catalytic triad The results above suggested that mutations in catalytic triad could abolish the activity of TSP50 to promote cell growth in vitro, and our
2.15. Statistical analysis Experiments were repeated at least three times with two replicates per sample for each experiment. Luciferase activity was normalized by β-galactosidase activity. The Student t-test was used to calculate the statistical significance of the experimental results. The significance level was set as *P b 0.05 and **P b 0.01. Error bars denote the standard deviations (SDs).
3. Results 3.1. Acquisition of cell strains stably expressing TSP50 and its point mutants Previous results have suggested that TSP50 exhibited enzyme activity and the threonine catalytic site mutation in the catalytic triad lead to loss of its protease activity [15]. To determine whether the catalytic triad is essential for protease activity and proliferationpromoting function of TSP50, we firstly established three point mutant constructs in the catalytic triad, named TSP50 T310A, TSP50 D206A and TSP50 H153A respectively, and the mutations were confirmed by sequencing. Then, the plasmids carrying a wild type TSP50 gene and the different TSP50 mutants were transfected into CHO cells to examine the effects of point mutations on TSP50-mediated cell growth promotion. After G418 pressure selection, stable transfected cell lines were obtained. Equal expression levels of TSP50 and the mutants were confirmed by RT-PCR and western blotting analysis (Fig. 1A and B), indicating that these cell strains with stable expression of TSP50 and its point mutants were available for further functional analysis.
Fig. 1. Acquisition of the point mutation constructs in the catalytic triad of TSP50 and analysis of the TSP50 protein expression in cell lines stably transfected with these plasmids. (A) TSP50 mRNA levels in TSP50 and the three TSP50 mutants-expressing cells were analyzed by RT-PCR. β-actin was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. (B) TSP50 protein levels in TSP50 and the three TSP50 mutant-expressing cells were analyzed by Western blotting. GAPDH was used as a loading control.
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Fig. 2. Abolishment of TSP50-mediated promotion of cell growth by its point mutations in catalytic triad. (A) Effects of point mutations in the catalytic triad on TSP50-mediated cell proliferation determined by MTT assay. The data are shown as the mean ± SEM of three separate experiments. *indicates P b 0.05 with respect to the value for mutant cells. (B) Effects of point mutations in the catalytic triad on TSP50-mediated cell proliferation determined by BrdU incorporation assay. Results are representative of three independent experiments. Statistical analysis was performed using the Student's t test. *indicates P b 0.05 with respect to the value for mutant cells. (C) Effects of point mutations in the catalytic triad on TSP50-mediated colony formation ability determined by soft agar colony formation assay. (D) The number of colonies with diameters over 1 mm in each dish was determined.*indicates P b 0.05 with respect to the value for mutant cells. (E) The amount of ki-67 was evaluated by immunofluorescence assay in TSP50 and mutant-expressing cells. Results are from one experiment that was representative of three independent experiments. Scale bar: 50 μm. (F) Ki67-positive signals was measured and quantified by image J software. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
prior studies have shown that TSP50-mediated tumorigenicity could be abolished by the TSP50 T310A point mutation [16]. We next examine whether the two other residues in catalytic triad also affect the tumorigenicity of TSP50 in vivo (TSP50 T310A mutation is a positive control here). Following the dorsal subcutaneous injection of 5 × 106 cells from each stable cell line expressing either wild type or different TSP50 mutants into athymic male nude mice, tumor growth was assessed. As shown in Fig. 3A and B, compared to control, the growth rate of tumors derived from TSP50-expressing cells was much faster; most importantly, tumor sizes were dramatically reduced in the animals injected with three TSP50 mutants-expressing cells. Furthermore, tumors derived from TSP50 D206A-expressing cells were lighter than those from two other mutants-expressing cells (Fig. 3C and D). Next, tumors from each group were subjected to immunohistochemical analysis with antibodies against Ki67. We found that TSP50-mediated hyperproliferation is impaired in tumors derived from cells expressing three different TSP50 mutants (Fig. 3E and F). These data indicate that the catalytic triad of TSP50, especially aspartic acid at 206, was required for TSP50-mediated tumorigenicity.
3.4. Abolishment protease activity of TSP50 by point mutations in its catalytic triad Next, we clarified why point mutations in catalytic triad of TSP50 could impair its proliferation-promoting function. Previous results shown that TSP50 was located in the ER (endoplasmic reticulum) and cytoplasmic membrane [15], so we first test whether there had been any change in cellular localization of the mutant TSP50s. The cellular localization of TSP50 and its three mutants was examined by immunofluorescent staining and western blotting with anti-TSP50 antibodies, and the results showed that TSP50 and its three mutants showed similar cellular localization (Fig. 4A and B), suggesting that the mutations in its catalytic triad didn't change the cellular localization of TSP50. TSP50 had been exhibited a better cleavage efficiency for β-casein and no enzyme activity was observed when threonine at position 310 was replaced by alanine [15]. Then, we purified the TSP50 proteins expressed by the plasmids carrying a wild type TSP50 gene and the different TSP50 mutants via the TSP50 antibody-affinity column, and evaluated the effects of two other residues in its catalytic triad on its protease activity
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Fig. 3. Effects of point mutations in the catalytic triad on TSP50-mediated tumorigenicity. (A) Panel showing tumor-bearing mice injected with indicated cells at day 16 postinjection. Cells were subcutaneously injected into recipient mice. After 16 days, the mice were sacrificed and all tumors were dissected. (B) Appearance of tumors deriving from each group. (C and D) Average tumour volume and tumor weight of each group 16 days after injection. Statistical analysis was performed using Student's t test. (E) Immunohistochemical analysis of Ki67 expression in histological sections of tumors from each group. Results are from one experiment that was representative of three independent experiments. Scale bar: 50 μm. (F) Quantitative analysis of Ki67 expression in histological sections of tumors from each group by image J software. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
by zymogram gel assays using β-casein as a substrate. Our studies showed that the wild type TSP50 protein, but not its mutants, exhibited a cleavage of β-casein (Fig. 4C and D), indicating that mutations in the catalytic triad of TSP50 lead to loss of its protease activity.
3.5. Effects of point mutations in catalytic triad on enhancement of NF-κB signaling by TSP50 NF-κB activation requires the phosphorylation, ubiquitylation and then degradation of IκBα, which allows the nuclear translocation of NF-κB dimmers [26]. Previous studies have shown that TSP50 could activate NF-κB signaling by enhancing IκBα degradation and increasing p65 nuclear translocation [20]. Based on the results that point mutations in catalytic triad of TSP50 can impair its proliferation-promoting function and protease activity, and TSP50-mediated NF-κB activation could be suppressed by the TSP50 T310A point mutation, we next determined whether two other mutant TSP50s could abolish TSP50mediated activation of NF-κB signaling. As shown in Fig. 5A, mutations in the catalytic triad, especially aspartic acid mutation at 206, effectively
inhibited the NF-κB-responsive luciferase reporter activity induced by TSP50. Furthermore, the degradation of IκBα in different TSP50 mutants-expressing cells was delayed (Fig. 5B), and the translocation of p65 to the nucleus was decelerated in the mutants-expressing cell, especially in TSP50 D206A-expressing cells, compared with wild-type TSP50 cells as demonstrated by Western blotting (Fig. 5C) and immunofluorescence detection (Fig. 5D and E). These observations provided strong evidences that point mutations in catalytic triad could impair the enhancement of NF-κB signaling by TSP50.
3.6. Effects of point mutations in catalytic triad on elevated expression of NF-κB-dependent gene products NF-κB regulated the expression of several genes whose products are involved in cell proliferation and apoptosis, and we have shown that TSP50 could significantly elevate the expression of NF-κB-dependent gene products [20,27]. Therefore, we investigated whether two other TSP50 mutants affected the expression of these proteins. As shown in Fig. 6A and B, overexpression of TSP50 dramatically enhanced the
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Fig. 4. Effects of point mutations in the catalytic triad on cellular localization and protease activity of TSP50. (A) Cellular localization of TSP50 and mutant TSP50s were determined by immunofluorescent staining with anti-TSP50 antibodies. Scale bar: 10 μm. (B) The levels and localization of TSP50 and its three mutants in the cytoplasm were measured by Western blotting and quantified by image J software. Top: the expression levels of TSP50 and its three mutants in the cytoplasm were examined by Western blotting. Bottom: quantitative analysis of Western blotting data by image J software. Values correspond to the mean ± S.E. of three independent experiments. (C) Top: The effects of point mutations in the catalytic triad on its protease activity were examined by zymogram gel assays using β-casein as a substrate. The white stripes in the third lane indicated the cleavage of β-casein. Bottom: quantitative analysis of zymogram gel assays. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (D) A duplicate gel without the casein substrate for Western blotting analysis using the TSP50 antibody.
expression of proliferation-related proteins cyclin D1, Cox-2, C-myc, and anti-apoptotic protein Bcl-2. However, overexpression of the three TSP50 mutants failed to enhance the expression of these NF-kB target genes, suggesting that the catalytic triad of TSP50 is essential for NF-κB-dependent gene expression. 3.7. Abolishment of the binding and degradation of IκBα by TSP50 Our previous studies have shown that TSP50 could activate NF-κB signaling pathway through interacting with NF-κB:IκBα complex and increasing degradation of IκBα, which could be one of the mechanisms underlying TSP50-mediated promotion of cell proliferation. To determine whether TSP50 mutants can interact with NF-κB: IκBα complex, co-immunoprecipitation experiments were performed. Consistent with our previous studies, TSP50 T310A mutants were not found to interact with the NF-κB:IκBα complex [16]; most importantly, we showed that TSP50 D206A and TSP50 H153A mutants also lost the ability to associate with the NF-κB:IκBα complex under the same conditions as wild-type molecules (Fig. 7A and B). In addition, a reciprocal immunoprecipitation/Western blotting experiment using the anti-IκBα antibody also showed that IκBα coprecipitated with
wild-type TSP50, but not the three TSP50mutants (Fig. 7C and D), which further confirmed the abolished interaction between different TSP50 mutants and IκBα. Based on the observation that TSP50 could bind to the NF-κB:IκBα complex and promote the degradation of IκBα proteins, we hypothesized that TSP50 could interact with IκBα directly and cleave it as a protease. To test this hypothesis, we first acquired the TSP50: IκBα proteins complex by passing the cell lysates from wild-type TSP50 through the TSP50 antibody-affinity column and eluting it with elution buffer in immunoprecipitation kit. Next, we incubated the TSP50: IκBα proteins complex in 20 mmol/L Tris–HCl (pH 7.6) at 37 °C for an hour to recover the protease activity of TSP50. Then, Western blotting assay was performed to examine the IκBα protein level. As expected, IκBα proteins in the complex decreased greatly due to the cleavage by wild-type TSP50 (Fig. 7E and F). To further confirm that the decrease of IκBα in cell extracts was due to TSP50 protease cleavage, protease inhibitor PMSF was added. As shown in Fig. 7G and H, PMSF effectively reversed the IκBα level decreased by TSP50. Collectively, our results revealed that TSP50, as a protease, could interact with and cleave IκBα protein directly, and the integrity of the catalytic triad of TSP50 is essential for an efficient degradation of IκBα and activating NF-kB signaling pathway.
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Fig. 5. Point mutations in the catalytic triad decreases TSP50's ability to promote NF-kB signaling pathway. (A) Effects of point mutations on NF-κB-dependent reporter gene expression. HEK293T cells were transiently transfected with pNF-κB-luc along with pcDNA3-TSP50 or different mutant TSP50s, 48 h after transfection, cells were pretreated with 100 ng/mL of PMA for 15 min and luciferase activities were assayed. *indicates P b 0.05 with respect to the value for mutant cells. (B) Effects of TSP50 or mutant TSP50s on IκBα degradation. Top: Indicated cells were treated with 100 ng/mL of PMA for 15 min, and then IκBα protein levels in these cells were analyzed by Western bolting. Bottom: quantitative analysis of Western blotting data by image J software. Levels of the IκBα were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (C) Top: the p65 nuclear localization was detected by Western blotting. Bottom: quantitative analysis of Western blotting data in the nucleus by image J software. Levels of the p65 were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (D) The p65 nuclear localization was detected by immunofluorescence assay. Scale bar: 10 μm. (E) Colocalization of P65 and DAPI was quantified by BD pathway 855 (Attovision software). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
4. Discussion Almost one-third of all proteases can be classified as serine proteases and they are implicated in many important processes such as digestion, inflammation, blood clotting and immune response [28]. TSP50 has a similar catalytic structure to many serine proteases, it contains His and Asp, at position 153 and 206 respectively in the catalytic triad, while
the Ser, at position 310, which the serine protease nomenclature is based on, was replaced by threonine, which made it a unique protease candidate [18,19]. Our previous results revealed that TSP50 could activate NF-κB signaling pathway through interaction with NF-κB: IκBα complex and degradation of IκBα proteins, and threonine mutation in the catalytic triad significantly depressed TSP50-induced cell proliferation and tumor formation [16,20].
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Fig. 6. Effects of point mutations in the catalytic triad on the expression of NF-kB target genes. (A) Left: different cells were treated with PMA (100 ng/mL) for 4 h, and then the expression of cyclin D1, Cox-2 and C-myc in cytoplasmic extracts was analyzed by Western blotting using indicated antibodies. Right: quantitative analysis of Western blotting data by image J software. Levels of these proteins were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (B) Left: effects of point mutations in the catalytic triad on expression of Bcl-2. Right: quantitative analysis of Western blot data by image J software. Levels of Bcl-2 were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
It has been shown that the three residues in the active site in chymotrypsin are distributed throughout the 240 amino acid residues, but are located in close proximity in the active three-dimensional conformation of the enzyme; furthermore, when all three of the catalytic triad residues are changed to other residues, they can retain some ability to catalyze the reaction, and increase the rate of hydrolysis by about 5 × 104-fold over the uncatalyzed reaction [5,7]. Increasing evidences have suggested that Asp in the catalytic triad may be involved in the stabilization of the ion-pair generated between the imidazolium ion and the negatively charged-tetrahedral intermediate, and Asp may participate in the orientation of the correct tautomer of His relative to Ser in the catalytic triad [29–32]. Here, we found that point mutations in the catalytic triad greatly abolished TSP50-mediated cell-proliferation and tumorigenicity, suggesting that the three residues in the catalytic triad were required for TSP50-mediated cell growth and tumorigenicity; most importantly, the Asp at position 206 may play a crucial role in maintaining the function of TSP50 (Figs. 2, 3). Interestingly, we found that the D206A mutant appeared to generate smaller tumors than the control, indicating that the Asp at position 206 plays a much more important role in maintaining the function of TSP50 in vivo than in vitro and further studies were needed to clarify the issue. Previous results have suggested that certain serine proteases function as signaling molecules to specifically regulate target cells by cleaving and triggering members of a new family of proteinaseactivated receptors (PARs) that are expressed in a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation [4]. For example, thrombin, a serine protease, is mitogenic in many cells due to activation of PAR-1 and subsequent activation of MAP kinases, and trypsin cleaves PAR-2, exposing a tethered ligand that
binds and activates the cleaved receptor [33,34]. This defines novel functions for serine proteases. In the present study, we found that point mutations in the catalytic triad could impair the enhancement of IκBα degradation and p65 nuclear translocation induced by TSP50; in addition, the increased expression of NF-kB target genes was also impaired by point mutations (Figs. 5, 6). These results imply that the integrity of the three residues in the catalytic triad is required for the activation of NF-κB signaling. The catalytic action of serine peptidases depends on the interplay of a nucleophile, a general base and an acid, the Asp draws protons on the His, which in turn pulls on protons on the ser; this tug-a-war like mechanism makes the ser nucleophilic, so that it can react with the protein and cause cleaving of the bond [7,35]. To determine whether TSP50 function as a protease in NF-κB-mediated cell growth, we examined the cleavage efficiency of TSP50 for IκBα protein. We found that TSP50, as a protease, can interact with IκBα protein and cleave it effectively. In contrast, mutant TSP50s can't bind to IκBα and cleave it (Fig. 7), suggesting that the integrity of the catalytic triad of TSP50 is required for its ability to cleave IκBα protein. However, whether TSP50 can modulate other proteins is an issue for further research. 5. Conclusions Our data highlight the importance of the catalytic triad, especially the aspartic acid 206, for TSP50-mediated cell proliferation and tumor formation. Furthermore, we found that the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. In addition, our results suggested that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Importantly, we found
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Fig. 7. Point mutations in the catalytic triad decreases TSP50's ability to bind and cleave IκBα protein. (A) Co-immunoprecipitation assays were performed to examine the interaction between TSP50 and mutant TSP50s with NF-kB:IκBα complex. Whole-cell lysates from different cells were immunoprecipitated with an anti-TSP50 antibody, followed by Western blotting (WB) with anti-IκBα antibodies. (B) Quantification of the co-immunoprecipitation data in (A). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (C) The results of a reciprocal immunoprecipitation/Western blotting experiment using anti-IκBα antibody, followed by western blotting assay with anti-TSP50 antibody. (D) Quantification of the co-immunoprecipitation data in (C). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (E) Acquisition of the TSP50:IκBα proteins complex from wild-type TSP50 was performed as described in Materials and methods, and the complex was incubated in 20 mmol/L Tris–HCl (pH 7.6) at 37 °C, then the IκBα protein level was evaluated by Western blotting assay. (F) Quantification of Western blotting data in (E). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05. (G) PMSF reversed the IκBα level decreased by TSP50. PMSF was mixed with the TSP50: IκBα proteins complex at a final concentration of 10 mmol/L, and IκBα protein level was evaluated by Western blotting assay. (H) Quantification of the Western blotting data in (G). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05.
that TSP50 promoted NF-κB signaling by interacting with IκBα and cleave it directly. Understanding the molecular mechanisms of TSP50induced tumorigenesis will provide novel anti-cancer strategies for treating humans. Authors' contributions YLB and YXL conceived and designed the experiments. ZBS, BL, PW and YYL performed the experiments. ZBS, YXH, YW and LGS analyzed the data. ZBS, CLY, YS and LHZ wrote the paper. All authors read, commented and approved the final manuscript. Conflict of interest statement The authors declared no conflict of interest. Acknowledgments The National Natural Science Foundation of China (Grant Nos. 81272919 and 81272242); the China Postdoctoral Science Foundation
(Grant No. 20090451122); the National Key New Drug Creation and Manufacturing Program of Ministry of Science and Technology (Grant No. 2011ZX09401-305-03);the National High Technology Research and Development Program of China (No. 2012AA02A407) the Scientific and Technological Developing Scheme of Ji Lin Province (Grant No. 20140520004JH); and the Fundamental Research Funds for the Central Universities (Grant No. 14QNJJ015). References [1] N.D. Rawlings, A.J. Barrett, Biochem. J. 290 (Pt 1) (1993) 205–218. [2] A.J. Barrett, N.D. Rawlings, Arch. Biochem. Biophys. 318 (1995) 247–250. [3] H.C. Cheng, M. Abdel-Ghany, R.C. Elble, B.U. Pauli, J. Biol. Chem. 273 (1998) 24207–24215. [4] O. Dery, C.U. Corvera, M. Steinhoff, N.W. Bunnett, Am. J. Physiol. 274 (1998) C1429–C1452. [5] M.M. Krem, E. Di Cera, EMBO J. 20 (2001) 3036–3045. [6] J.D. Cheng, R.L. Dunbrack Jr., M. Valianou, A. Rogatko, R.K. Alpaugh, L.M. Weiner, Cancer Res. 62 (2002) 4767–4772. [7] L. Hedstrom, Chem. Rev. 102 (2002) 4501–4524. [8] R.J. Grand, A.S. Turnell, P.W. Grabham, Biochem. J. 313 (Pt 2) (1996) 353–368. [9] M.D. Hollenberg, Trends Pharmacol. Sci. 17 (1996) 3–6. [10] L.F. Brass, M. Molino, Thromb. Haemost. 78 (1997) 234–241.
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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
The catalytic triad of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation Zhen Bo Song a,b, Biao Liu a,c,d, Yu Yin Li a,b, Ping Wu b, Yong Li Bao a,⁎, Yan Xin Huang b,c, Yin Wu b, Lu Guo Sun a,b, Chun Lei Yu a,b, Ying Sun c, Li Hua Zheng c, Yu Xin Li b,c,⁎ a
National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun 130024, China Research Center of Agriculture and Medicine Gene Engineering of Ministry of Education, Northeast Normal University, Changchun 130024, China Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China d Department of Handsurgery, China–Japan Union Hospital, Jilin University, Changchun 130033, China b c
a r t i c l e
i n f o
Article history: Received 14 May 2014 Received in revised form 22 June 2014 Accepted 4 July 2014 Available online 15 July 2014 Keywords: TSP50 Serine protease Catalytic triad Mutants Cell proliferation Mechanism
a b s t r a c t Testes-specific protease 50 (TSP50) is a novelly identified pro-oncogene and it shares a similar enzymatic structure with many serine proteases. Our previous results suggested that TSP50 could promote tumorigenesis through degradation of IκBα protein and activating NF-κB signaling, and the threonine mutation in its catalytic triad could depress TSP50-mediated cell proliferation. However, whether the two other residues in the catalytic triad of TSP50 play a role in maintaining protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. Here, we constructed and characterized three catalytic triad mutants of TSP50 and found that all the mutants could significantly depress TSP50-induced cell proliferation and colony formation in vitro and tumor formation in vivo, and the aspartic acid at position 206 in the catalytic triad played a more crucial role than threonine and histidine in this process. Mechanistic studies revealed that the mutants in the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. Furthermore, our data indicated that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Most importantly, we demonstrated that TSP50 could interact with IκBα protein and cleave it directly as a new protease in vitro. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Serine proteases contain a large family of enzymes that are characterized by possessing an active serine in their catalytic site, and they play a critical role in numerous physiologic processes including digestion, blood coagulation, complement activation, fibrinolysis, reproduction, embryonic development, protein processing and tissue remodeling [1–6]. Serine proteases maintain a strictly conserved active site geometry among their catalytic Ser, His and Asp residues; this
Abbreviations: Bcl-2, B cell lymphoma/leukemia-2; BrdU, 5-bromo-2′-deoxyuridine; CHO cell, Chinese-hamster ovary cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK 293T, human embryonic kidney 293T cells; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB, nuclear factor κB; PMSF, phenylmethanesulphonyl fluoride; PMA, phorbol myristate acetate; RT-PCR, reverse transcription-PCR;TSP50, testes-specific protease 50. ⁎ Corresponding authors. Tel.: +86 431 8509 8455; fax: +86 431 8916 5917. E-mail addresses: [email protected] (Z.B. Song), [email protected] (B. Liu), [email protected] (Y.Y. Li), [email protected] (P. Wu), [email protected] (Y.L. Bao), [email protected] (Y.X. Huang), [email protected] (Y. Wu), [email protected] (L.G. Sun), [email protected] (C.L. Yu), [email protected] (Y. Sun), [email protected] (L.H. Zheng), [email protected] (Y.X. Li).
http://dx.doi.org/10.1016/j.cellsig.2014.07.012 0898-6568/© 2014 Elsevier Inc. All rights reserved.
shared catalytic structure suggests that common architectural motifs are likely to be found in the molecular designs of active sites utilizing a Ser-His-Asp triad [5]. The substitution of either His57 or Ser195 to Ala is sufficient to completely disable the catalytic triad, and the substitution of Asp102 to Asn in trypsin decreases kcat/Km by 104 at neutral pH [7]. Increasing evidences indicates that some serine proteases, which have been traditionally viewed as degradative enzymes, are also signaling molecules that regulate multiple cellular functions by activating specific receptors [8–10], protease receptors such as PAR-1, PAR-3 and PAR-2, were cleaved and activated by thrombin and trypsin-like enzymes respectively [11–13]. TSP50 is a testis-specific gene encoding a protein that is homologous to serine proteases, and it shares two critical catalytic triads, histidine and aspartic acid at positions 153 and 206, respectively. However, the most crucial triad, serine, at position 310 was replaced by threonine and it possessed enzymatic activity [14,15], suggesting that TSP50 might represent a novel type of protease. When the catalytic threonine residual of TSP50 is replaced by alanine, it was unable to cleave β-casein [15]. Our recent results showed that TSP50 T310A mutation significantly depressed TSP50-induced cell proliferation, colony formation, resisting apoptosis, cell migration and cell invasion in vitro and abolished the oncogenicity of TSP50 in vivo [16]. These results strongly indicated
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that the threonine in the catalytic triad of TSP50 is essential to its function in tumorigenesis. However, whether this is correlated to protease activity of TSP50, the role of the two other residues in catalytic triad of TSP50 in protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. In addition to expression in normal testes, TSP50 was abnormally expressed at high level in many breast cancer and colorectal carcinoma biopsies tested [17–19]. Previously, we revealed that overexpression of TSP50 efficiently promotes cell proliferation and tumor formation through the activation of the NF-κB signaling pathway, and TSP50 can promote the degradation of κBα proteins by binding to the NF-κB: IκBα complex [20]. However, whether TSP50 can interact with IκBα directly and cleave it as a protease is unknown. In this paper, we mainly studied the effects of the histidine and aspartic acid mutation in the catalytic triad of TSP50 on its function in tumorigenesis and the mechanisms involved this process. We provided evidences that the mutations in the catalytic triad of TSP50 could significantly depressed TSP50-mediated cell proliferation and tumor formation, and aspartic acid at positions 206 played a much more important role in maintaining the function of TSP50.
using LipofectamineTM2000 (Invitrogen), according to the instructions of the manufacturer. Transfected cells were incubated in the presence of G418 for 2 weeks and the stable transfected cell lines were selected.
2. Materials and methods
Cytosolic and nuclear extracts were prepared and Western blotting was performed as described previously [22].
2.4. RNA extract and RT-PCR Total RNA was prepared from cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The RT-PCR kit was bought from transGgenbiotech. Total RNA (3 μg) was reverse transcribed into cDNA by incubating at 50 °C for 60 min. TSP50 mRNA was amplified from cDNA templates by RT-PCR using the primers 5′-CGGATCCATGCAGGGGAAGCC-3′ (sense) and 5′-GCTCTAGAAGTCAGAGGGCAG-3′ (antisense), β-actin primers 5′-TCGTGCGTGACATTAAGGAG-3′ (sense) and 5′-ATGCCAGGGTACAT GGTGGT-3′ (antisense). PCR was performed for 30 cycles (each cycle consisting of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s). The PCR products were analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide and visualized under UV light. 2.5. Western blotting assay
2.1. Antibodies and reagents 2.6. MTT assay Polyclonal antibodies against p65, IκBα, Histone1, C-Myc, COX-2, cyclinD1, Ki-67, β-actin and Bcl-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody against GAPDH was purchased from Kangcheng Biotech (Shanghai, China). Anti-TSP50 monoclonal antibody was prepared in our laboratory [21]. The site-directed Gene Mutagenesis Kit and the Calcium Phosphate Cell Transfection Kit were purchased from Beyotime (Shanghai, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The 5-bromo-2′-deoxyuridine (BrdU) labeling and detection ELISA kit was purchased from Roche Diagnostics (Mannheim, Germany). The Pierce Crosslink IP Kit was bought from Thermo Scientific (no: 26147).
Cell growth was determined by MTT assay. The cells were plated at 1 × 104 cells per well in 96-well microtiter plates. After incubation for the indicated time, MTT (5 mg/mL) was added to each well and incubated for 4 h. The absorbance was recorded on a microplate reader at a wavelength of 570 nm. 2.7. Colony formation in soft agar One milliliter base agar (1.2% agar plus 2 × DMEM in equal volumes) was added to six-well plates. Twenty four hours prior to initiating the soft agar assay, 4 × 103 cells/well were added in 1 mL of top agar (0.7% agar mixed with 2× DMEM in equal volumes) to each well. Plates were maintained at 37 °C for 14 days before colonies were counted.
2.2. Cell lines and cell culture 2.8. BrdU incorporation assay CHO cells (Chinese hamster ovary cells) and HEK 293T cells (human embryonic kidney 293T cells) were obtained from the Chinese Academy of Sciences Shanghai Institute for Biological Sciences-Cell Resource Center, which had characterized the cell lines by short tandem repeat profiling, cell morphology and karyotyping assay. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen, USA), which was supplemented with 10% fetal bovine serum (TBD Science, Tianjin, China), 100 U/mL penicillin and 100 μg/mL streptomycin (Ameresco, USA), at 37°C with 5% CO2.
DNA synthesis was assessed by measuring the incorporation of BrdU into newly synthesized strands. Cells were replated at 1 × 103 cells/well on 96-well plates. Twenty-four hours after plating, BrdU labeling was initiated by adding the labeling solution at a final concentration of 10 μM to the culture medium. After the cells were incubated for 6 h, labeling was stopped and BrdU uptake was measured according to the protocol of the manufacturer. 2.9. Analysis of luciferase activity
2.3. Plasmid constructs and transfection The EGFP fusion protein plasmids and the pNF-κB-luc plasmid were prepared in our laboratory. The three point mutation constructs of TSP50, T310A, D206A and H153A originated from the construct pcDNA3.0-TSP50 were achieved using Sit Directed Mutagenesis Kit. The primer sequences used were as follows: TSP50 T310A-For: 5′-GTTCTGCTATGAGCTAGCTGGAGAGCCCTTGGTC-3′, Rev: 5′-GACCAA GGGCTCTCCAGCTAGCTCATAGCAGAAC-3′, TSP50 D206A-For: 5′-GTGG GCCAGGCCAACGCCATCGGCCTCCTCAAG-3′, Rev: 5′-CTTGAGGAGGCC GATGGCGTTGGCCTGGCCCAC-3′, TSP50 H153A -For: 5′-GGTGCTGACT GTGGCCGCCTGCCTGATCTGGCGTG-3′, Rev: 5′-CACGCCAGATCAGGCA GGCGGCCACAGTCAGCACC-3′. The TSP50 and its point mutation constructs were cloned into the pcDNA3.0 basic vector. Cells were transfected with the T310A, D206A, and H153A plasmids per well
Firefly luciferase activity was measured 48 h after transfection. Analysis of luciferase activity was performed as described previously [23]. 2.10. Immunofluorescence detection The localization of p65 and Ki-67 staining were carried out in accordance with previously described procedures [24]. 2.11. Co-immunoprecipitation Co-immunoprecipitation experiments were performed by using the Pierce Crosslink Immunoprecipitation kit from Thermo Scientific according to the standard protocol of the manufacturer.
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2.12. Protein purification To purify endogenous TSP50 and its three mutants, TSP50 antibodyaffinity column was prepared by binding of antibody to protein A/G plus agarose and the bound antibody was crosslinked by the DSS crosslinker following the manufacturer's instructions. TSP50 was purified by passing the cell lysate through the TSP50 antibody-affinity column and eluting it with elution buffer provided in immunorecipitation kit.
2.13. Zymogram gel assay To test enzymatic activity, equal amounts of TSP50 and mutant TSP50 proteins were applied in duplicate to 12% Tris–glycine gels containing 1 mg/mL β-casein. After electrophoresis, the gels were washed with 2.5% Triton X-100 twice for 30 min, rinsed three times for 30 min with a 50 mM Tris–HCl buffer (pH 7.6) containing 5 mM CaCl2, 0.02% Brij-35, and 0.2% sodium azide, and then incubated overnight at 37 °C. After the enzymatic reactions, the gels were stained with Coomassie blue R-250 and destained with acetic acid and methanol. Areas of enzyme activity were detected as clear bands against the blue-stained background. To normalize the enzyme digested band created by TSP50, duplicate cell extracts were prepared for Western analysis. The experiments were done three times.
2.14. Tumorigenicity studies and immunohistochemistry assay Male BALB/c nude mice (6-weeks old) were purchased from the Chinese Academy of Sciences and were housed under pathogenfree conditions. Analysis of tumorigenicity and immunohistochemistry were performed as described previously [16]. Animal experiments were performed in accordance with established guidelines. The Chinese Academy of Sciences Animal Care and Use Committee gave approval for the animal experiments.
3.2. Effects of point mutations in catalytic triad on TSP50-mediated promotion of cell growth Previously, our results have shown that cell proliferation and colony formation were significantly promoted by overexpression of TSP50 in vitro [20,25], and TSP50 T310A mutation severely restrained cell proliferation [16]. Here, we mainly evaluated the effects of two other residues (Histidine153 and Aspartate206) in catalytic triad on TSP50mediated cell proliferation, and compared the function differences among the different TSP50 mutants (TSP50 T310A mutation is a positive control here). Results from the MTT cell proliferation assay demonstrated that expression of TSP50 T310A, TSP50 D206A and TSP50 H153A greatly destroyed the cell-proliferation-promoting activity by expression of TSP50. What is more, TSP50 D206A-expressing cells grew most slowly (Fig. 2A). Similarly, when DNA synthesis was measured by BrdU incorporation, we found that the three TSP50 mutants greatly decreased the uptake of BrdU in different degrees (Fig. 2B). Moreover, the anchorage-independent growth ability of TSP50 and the mutantexpressing cells was assayed by colony formation in soft agar, where the number of colonies obtained in the TSP50 mutant-expressing cultures was significantly lower than the number obtained in the TSP50-expressing cells; furthermore, TSP50 D206A-expressing cells formed much less colonies than those by two other mutants (Fig. 2C and D). Antigen Ki-67 is a nuclear protein and it is strictly associated with cell proliferation. Fluorescence assay suggested that the three TSP50 mutants strongly impaired the expression of Ki-67 induced by TSP50 (Fig. 2E and F). Overall, our results suggested that all three residues in the catalytic triad were indispensable for TSP50-mediated cell growth, and aspartic acid at position 206 may play a crucial role. 3.3. Abolishment of TSP50-mediated tumorigenicity by point mutations in its catalytic triad The results above suggested that mutations in catalytic triad could abolish the activity of TSP50 to promote cell growth in vitro, and our
2.15. Statistical analysis Experiments were repeated at least three times with two replicates per sample for each experiment. Luciferase activity was normalized by β-galactosidase activity. The Student t-test was used to calculate the statistical significance of the experimental results. The significance level was set as *P b 0.05 and **P b 0.01. Error bars denote the standard deviations (SDs).
3. Results 3.1. Acquisition of cell strains stably expressing TSP50 and its point mutants Previous results have suggested that TSP50 exhibited enzyme activity and the threonine catalytic site mutation in the catalytic triad lead to loss of its protease activity [15]. To determine whether the catalytic triad is essential for protease activity and proliferationpromoting function of TSP50, we firstly established three point mutant constructs in the catalytic triad, named TSP50 T310A, TSP50 D206A and TSP50 H153A respectively, and the mutations were confirmed by sequencing. Then, the plasmids carrying a wild type TSP50 gene and the different TSP50 mutants were transfected into CHO cells to examine the effects of point mutations on TSP50-mediated cell growth promotion. After G418 pressure selection, stable transfected cell lines were obtained. Equal expression levels of TSP50 and the mutants were confirmed by RT-PCR and western blotting analysis (Fig. 1A and B), indicating that these cell strains with stable expression of TSP50 and its point mutants were available for further functional analysis.
Fig. 1. Acquisition of the point mutation constructs in the catalytic triad of TSP50 and analysis of the TSP50 protein expression in cell lines stably transfected with these plasmids. (A) TSP50 mRNA levels in TSP50 and the three TSP50 mutants-expressing cells were analyzed by RT-PCR. β-actin was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. (B) TSP50 protein levels in TSP50 and the three TSP50 mutant-expressing cells were analyzed by Western blotting. GAPDH was used as a loading control.
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Fig. 2. Abolishment of TSP50-mediated promotion of cell growth by its point mutations in catalytic triad. (A) Effects of point mutations in the catalytic triad on TSP50-mediated cell proliferation determined by MTT assay. The data are shown as the mean ± SEM of three separate experiments. *indicates P b 0.05 with respect to the value for mutant cells. (B) Effects of point mutations in the catalytic triad on TSP50-mediated cell proliferation determined by BrdU incorporation assay. Results are representative of three independent experiments. Statistical analysis was performed using the Student's t test. *indicates P b 0.05 with respect to the value for mutant cells. (C) Effects of point mutations in the catalytic triad on TSP50-mediated colony formation ability determined by soft agar colony formation assay. (D) The number of colonies with diameters over 1 mm in each dish was determined.*indicates P b 0.05 with respect to the value for mutant cells. (E) The amount of ki-67 was evaluated by immunofluorescence assay in TSP50 and mutant-expressing cells. Results are from one experiment that was representative of three independent experiments. Scale bar: 50 μm. (F) Ki67-positive signals was measured and quantified by image J software. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
prior studies have shown that TSP50-mediated tumorigenicity could be abolished by the TSP50 T310A point mutation [16]. We next examine whether the two other residues in catalytic triad also affect the tumorigenicity of TSP50 in vivo (TSP50 T310A mutation is a positive control here). Following the dorsal subcutaneous injection of 5 × 106 cells from each stable cell line expressing either wild type or different TSP50 mutants into athymic male nude mice, tumor growth was assessed. As shown in Fig. 3A and B, compared to control, the growth rate of tumors derived from TSP50-expressing cells was much faster; most importantly, tumor sizes were dramatically reduced in the animals injected with three TSP50 mutants-expressing cells. Furthermore, tumors derived from TSP50 D206A-expressing cells were lighter than those from two other mutants-expressing cells (Fig. 3C and D). Next, tumors from each group were subjected to immunohistochemical analysis with antibodies against Ki67. We found that TSP50-mediated hyperproliferation is impaired in tumors derived from cells expressing three different TSP50 mutants (Fig. 3E and F). These data indicate that the catalytic triad of TSP50, especially aspartic acid at 206, was required for TSP50-mediated tumorigenicity.
3.4. Abolishment protease activity of TSP50 by point mutations in its catalytic triad Next, we clarified why point mutations in catalytic triad of TSP50 could impair its proliferation-promoting function. Previous results shown that TSP50 was located in the ER (endoplasmic reticulum) and cytoplasmic membrane [15], so we first test whether there had been any change in cellular localization of the mutant TSP50s. The cellular localization of TSP50 and its three mutants was examined by immunofluorescent staining and western blotting with anti-TSP50 antibodies, and the results showed that TSP50 and its three mutants showed similar cellular localization (Fig. 4A and B), suggesting that the mutations in its catalytic triad didn't change the cellular localization of TSP50. TSP50 had been exhibited a better cleavage efficiency for β-casein and no enzyme activity was observed when threonine at position 310 was replaced by alanine [15]. Then, we purified the TSP50 proteins expressed by the plasmids carrying a wild type TSP50 gene and the different TSP50 mutants via the TSP50 antibody-affinity column, and evaluated the effects of two other residues in its catalytic triad on its protease activity
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Fig. 3. Effects of point mutations in the catalytic triad on TSP50-mediated tumorigenicity. (A) Panel showing tumor-bearing mice injected with indicated cells at day 16 postinjection. Cells were subcutaneously injected into recipient mice. After 16 days, the mice were sacrificed and all tumors were dissected. (B) Appearance of tumors deriving from each group. (C and D) Average tumour volume and tumor weight of each group 16 days after injection. Statistical analysis was performed using Student's t test. (E) Immunohistochemical analysis of Ki67 expression in histological sections of tumors from each group. Results are from one experiment that was representative of three independent experiments. Scale bar: 50 μm. (F) Quantitative analysis of Ki67 expression in histological sections of tumors from each group by image J software. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
by zymogram gel assays using β-casein as a substrate. Our studies showed that the wild type TSP50 protein, but not its mutants, exhibited a cleavage of β-casein (Fig. 4C and D), indicating that mutations in the catalytic triad of TSP50 lead to loss of its protease activity.
3.5. Effects of point mutations in catalytic triad on enhancement of NF-κB signaling by TSP50 NF-κB activation requires the phosphorylation, ubiquitylation and then degradation of IκBα, which allows the nuclear translocation of NF-κB dimmers [26]. Previous studies have shown that TSP50 could activate NF-κB signaling by enhancing IκBα degradation and increasing p65 nuclear translocation [20]. Based on the results that point mutations in catalytic triad of TSP50 can impair its proliferation-promoting function and protease activity, and TSP50-mediated NF-κB activation could be suppressed by the TSP50 T310A point mutation, we next determined whether two other mutant TSP50s could abolish TSP50mediated activation of NF-κB signaling. As shown in Fig. 5A, mutations in the catalytic triad, especially aspartic acid mutation at 206, effectively
inhibited the NF-κB-responsive luciferase reporter activity induced by TSP50. Furthermore, the degradation of IκBα in different TSP50 mutants-expressing cells was delayed (Fig. 5B), and the translocation of p65 to the nucleus was decelerated in the mutants-expressing cell, especially in TSP50 D206A-expressing cells, compared with wild-type TSP50 cells as demonstrated by Western blotting (Fig. 5C) and immunofluorescence detection (Fig. 5D and E). These observations provided strong evidences that point mutations in catalytic triad could impair the enhancement of NF-κB signaling by TSP50.
3.6. Effects of point mutations in catalytic triad on elevated expression of NF-κB-dependent gene products NF-κB regulated the expression of several genes whose products are involved in cell proliferation and apoptosis, and we have shown that TSP50 could significantly elevate the expression of NF-κB-dependent gene products [20,27]. Therefore, we investigated whether two other TSP50 mutants affected the expression of these proteins. As shown in Fig. 6A and B, overexpression of TSP50 dramatically enhanced the
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Fig. 4. Effects of point mutations in the catalytic triad on cellular localization and protease activity of TSP50. (A) Cellular localization of TSP50 and mutant TSP50s were determined by immunofluorescent staining with anti-TSP50 antibodies. Scale bar: 10 μm. (B) The levels and localization of TSP50 and its three mutants in the cytoplasm were measured by Western blotting and quantified by image J software. Top: the expression levels of TSP50 and its three mutants in the cytoplasm were examined by Western blotting. Bottom: quantitative analysis of Western blotting data by image J software. Values correspond to the mean ± S.E. of three independent experiments. (C) Top: The effects of point mutations in the catalytic triad on its protease activity were examined by zymogram gel assays using β-casein as a substrate. The white stripes in the third lane indicated the cleavage of β-casein. Bottom: quantitative analysis of zymogram gel assays. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (D) A duplicate gel without the casein substrate for Western blotting analysis using the TSP50 antibody.
expression of proliferation-related proteins cyclin D1, Cox-2, C-myc, and anti-apoptotic protein Bcl-2. However, overexpression of the three TSP50 mutants failed to enhance the expression of these NF-kB target genes, suggesting that the catalytic triad of TSP50 is essential for NF-κB-dependent gene expression. 3.7. Abolishment of the binding and degradation of IκBα by TSP50 Our previous studies have shown that TSP50 could activate NF-κB signaling pathway through interacting with NF-κB:IκBα complex and increasing degradation of IκBα, which could be one of the mechanisms underlying TSP50-mediated promotion of cell proliferation. To determine whether TSP50 mutants can interact with NF-κB: IκBα complex, co-immunoprecipitation experiments were performed. Consistent with our previous studies, TSP50 T310A mutants were not found to interact with the NF-κB:IκBα complex [16]; most importantly, we showed that TSP50 D206A and TSP50 H153A mutants also lost the ability to associate with the NF-κB:IκBα complex under the same conditions as wild-type molecules (Fig. 7A and B). In addition, a reciprocal immunoprecipitation/Western blotting experiment using the anti-IκBα antibody also showed that IκBα coprecipitated with
wild-type TSP50, but not the three TSP50mutants (Fig. 7C and D), which further confirmed the abolished interaction between different TSP50 mutants and IκBα. Based on the observation that TSP50 could bind to the NF-κB:IκBα complex and promote the degradation of IκBα proteins, we hypothesized that TSP50 could interact with IκBα directly and cleave it as a protease. To test this hypothesis, we first acquired the TSP50: IκBα proteins complex by passing the cell lysates from wild-type TSP50 through the TSP50 antibody-affinity column and eluting it with elution buffer in immunoprecipitation kit. Next, we incubated the TSP50: IκBα proteins complex in 20 mmol/L Tris–HCl (pH 7.6) at 37 °C for an hour to recover the protease activity of TSP50. Then, Western blotting assay was performed to examine the IκBα protein level. As expected, IκBα proteins in the complex decreased greatly due to the cleavage by wild-type TSP50 (Fig. 7E and F). To further confirm that the decrease of IκBα in cell extracts was due to TSP50 protease cleavage, protease inhibitor PMSF was added. As shown in Fig. 7G and H, PMSF effectively reversed the IκBα level decreased by TSP50. Collectively, our results revealed that TSP50, as a protease, could interact with and cleave IκBα protein directly, and the integrity of the catalytic triad of TSP50 is essential for an efficient degradation of IκBα and activating NF-kB signaling pathway.
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Fig. 5. Point mutations in the catalytic triad decreases TSP50's ability to promote NF-kB signaling pathway. (A) Effects of point mutations on NF-κB-dependent reporter gene expression. HEK293T cells were transiently transfected with pNF-κB-luc along with pcDNA3-TSP50 or different mutant TSP50s, 48 h after transfection, cells were pretreated with 100 ng/mL of PMA for 15 min and luciferase activities were assayed. *indicates P b 0.05 with respect to the value for mutant cells. (B) Effects of TSP50 or mutant TSP50s on IκBα degradation. Top: Indicated cells were treated with 100 ng/mL of PMA for 15 min, and then IκBα protein levels in these cells were analyzed by Western bolting. Bottom: quantitative analysis of Western blotting data by image J software. Levels of the IκBα were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (C) Top: the p65 nuclear localization was detected by Western blotting. Bottom: quantitative analysis of Western blotting data in the nucleus by image J software. Levels of the p65 were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (D) The p65 nuclear localization was detected by immunofluorescence assay. Scale bar: 10 μm. (E) Colocalization of P65 and DAPI was quantified by BD pathway 855 (Attovision software). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
4. Discussion Almost one-third of all proteases can be classified as serine proteases and they are implicated in many important processes such as digestion, inflammation, blood clotting and immune response [28]. TSP50 has a similar catalytic structure to many serine proteases, it contains His and Asp, at position 153 and 206 respectively in the catalytic triad, while
the Ser, at position 310, which the serine protease nomenclature is based on, was replaced by threonine, which made it a unique protease candidate [18,19]. Our previous results revealed that TSP50 could activate NF-κB signaling pathway through interaction with NF-κB: IκBα complex and degradation of IκBα proteins, and threonine mutation in the catalytic triad significantly depressed TSP50-induced cell proliferation and tumor formation [16,20].
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Fig. 6. Effects of point mutations in the catalytic triad on the expression of NF-kB target genes. (A) Left: different cells were treated with PMA (100 ng/mL) for 4 h, and then the expression of cyclin D1, Cox-2 and C-myc in cytoplasmic extracts was analyzed by Western blotting using indicated antibodies. Right: quantitative analysis of Western blotting data by image J software. Levels of these proteins were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (B) Left: effects of point mutations in the catalytic triad on expression of Bcl-2. Right: quantitative analysis of Western blot data by image J software. Levels of Bcl-2 were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells.
It has been shown that the three residues in the active site in chymotrypsin are distributed throughout the 240 amino acid residues, but are located in close proximity in the active three-dimensional conformation of the enzyme; furthermore, when all three of the catalytic triad residues are changed to other residues, they can retain some ability to catalyze the reaction, and increase the rate of hydrolysis by about 5 × 104-fold over the uncatalyzed reaction [5,7]. Increasing evidences have suggested that Asp in the catalytic triad may be involved in the stabilization of the ion-pair generated between the imidazolium ion and the negatively charged-tetrahedral intermediate, and Asp may participate in the orientation of the correct tautomer of His relative to Ser in the catalytic triad [29–32]. Here, we found that point mutations in the catalytic triad greatly abolished TSP50-mediated cell-proliferation and tumorigenicity, suggesting that the three residues in the catalytic triad were required for TSP50-mediated cell growth and tumorigenicity; most importantly, the Asp at position 206 may play a crucial role in maintaining the function of TSP50 (Figs. 2, 3). Interestingly, we found that the D206A mutant appeared to generate smaller tumors than the control, indicating that the Asp at position 206 plays a much more important role in maintaining the function of TSP50 in vivo than in vitro and further studies were needed to clarify the issue. Previous results have suggested that certain serine proteases function as signaling molecules to specifically regulate target cells by cleaving and triggering members of a new family of proteinaseactivated receptors (PARs) that are expressed in a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation [4]. For example, thrombin, a serine protease, is mitogenic in many cells due to activation of PAR-1 and subsequent activation of MAP kinases, and trypsin cleaves PAR-2, exposing a tethered ligand that
binds and activates the cleaved receptor [33,34]. This defines novel functions for serine proteases. In the present study, we found that point mutations in the catalytic triad could impair the enhancement of IκBα degradation and p65 nuclear translocation induced by TSP50; in addition, the increased expression of NF-kB target genes was also impaired by point mutations (Figs. 5, 6). These results imply that the integrity of the three residues in the catalytic triad is required for the activation of NF-κB signaling. The catalytic action of serine peptidases depends on the interplay of a nucleophile, a general base and an acid, the Asp draws protons on the His, which in turn pulls on protons on the ser; this tug-a-war like mechanism makes the ser nucleophilic, so that it can react with the protein and cause cleaving of the bond [7,35]. To determine whether TSP50 function as a protease in NF-κB-mediated cell growth, we examined the cleavage efficiency of TSP50 for IκBα protein. We found that TSP50, as a protease, can interact with IκBα protein and cleave it effectively. In contrast, mutant TSP50s can't bind to IκBα and cleave it (Fig. 7), suggesting that the integrity of the catalytic triad of TSP50 is required for its ability to cleave IκBα protein. However, whether TSP50 can modulate other proteins is an issue for further research. 5. Conclusions Our data highlight the importance of the catalytic triad, especially the aspartic acid 206, for TSP50-mediated cell proliferation and tumor formation. Furthermore, we found that the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. In addition, our results suggested that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Importantly, we found
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Fig. 7. Point mutations in the catalytic triad decreases TSP50's ability to bind and cleave IκBα protein. (A) Co-immunoprecipitation assays were performed to examine the interaction between TSP50 and mutant TSP50s with NF-kB:IκBα complex. Whole-cell lysates from different cells were immunoprecipitated with an anti-TSP50 antibody, followed by Western blotting (WB) with anti-IκBα antibodies. (B) Quantification of the co-immunoprecipitation data in (A). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (C) The results of a reciprocal immunoprecipitation/Western blotting experiment using anti-IκBα antibody, followed by western blotting assay with anti-TSP50 antibody. (D) Quantification of the co-immunoprecipitation data in (C). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05 with respect to the value for mutant cells. (E) Acquisition of the TSP50:IκBα proteins complex from wild-type TSP50 was performed as described in Materials and methods, and the complex was incubated in 20 mmol/L Tris–HCl (pH 7.6) at 37 °C, then the IκBα protein level was evaluated by Western blotting assay. (F) Quantification of Western blotting data in (E). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05. (G) PMSF reversed the IκBα level decreased by TSP50. PMSF was mixed with the TSP50: IκBα proteins complex at a final concentration of 10 mmol/L, and IκBα protein level was evaluated by Western blotting assay. (H) Quantification of the Western blotting data in (G). Values correspond to the mean ± S.E. of three independent experiments. *indicates P b 0.05.
that TSP50 promoted NF-κB signaling by interacting with IκBα and cleave it directly. Understanding the molecular mechanisms of TSP50induced tumorigenesis will provide novel anti-cancer strategies for treating humans. Authors' contributions YLB and YXL conceived and designed the experiments. ZBS, BL, PW and YYL performed the experiments. ZBS, YXH, YW and LGS analyzed the data. ZBS, CLY, YS and LHZ wrote the paper. All authors read, commented and approved the final manuscript. Conflict of interest statement The authors declared no conflict of interest. Acknowledgments The National Natural Science Foundation of China (Grant Nos. 81272919 and 81272242); the China Postdoctoral Science Foundation
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