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Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition
Biochemical and Biophysical Research Communications xxx (2016) 1e6
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition Hotaru Matsumoto a, Hisato Saitoh a, b, * a b
Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto, Japan Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2016 Accepted 3 May 2016 Available online xxx
We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. © 2016 Elsevier Inc. All rights reserved.
Keywords: Nuclear protein sequestration Puromycin Small ubiquitin-related modifier (SUMO) Ubiquitin proteasome system Promyelocytic leukemia nuclear bodies
1. Introduction Puromycin is an aminonucleoside antibiotic produced by Streptomyces alboniger [1]. When administrated to cells, it enters the ribosome A site, followed by incorporation into the nascent polypeptide chain, elongating either on the monoribosome or polyribosomes, which leads to covalent attachment of puromycin to the C-terminal end of the nascent polypeptides, referred to as ‘puromycylation’ [2e5]. Because puromycylation prematurely terminates elongation at any given site in the elongating polypeptide, the resulting puromycylated polypeptides are expected to be heterogeneous in size and improperly folded. Under standard conditions, such puromycylated polypeptides are subjected to an elimination reaction controlled by the ubiquitin-proteasome system (UPS) [6]. However, when the UPS is perturbed in cells, puromycylated immature polypeptides are expected to accumulate,
Abbreviations: SUMO, small ubiquitin-related modifier; PML-NBs, promyelocytic leukemia-nuclear bodies; RNF4, really interesting new gene (RING) finger protein 4; DAPI, 40 , 6-diamidino-2-phenylindole; OP-Puro, O-propargyl-puromycin. * Corresponding author. Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto, 860-8555, Japan. E-mail address: [email protected] (H. Saitoh).
causing cytotoxic effects, including stress response and growth defects, ultimately inducing cell death. To escape such a scenario, cells should contain an intrinsic protection system that would respond to accumulation of immature/aberrant proteins. However, the cellular defense strategy against immature/aberrant polypeptides remains largely unknown. The small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are highly conserved proteins, which can be covalently attached to multiple cellular proteins, referred to as SUMOylation [7,8]. Intriguingly, SUMOylation frequently, but not always, occurs with ubiquitin modification (ubiquitinylation) [9e12]. Many SUMOs and SUMOyalted proteins are detected in promyelocytic leukemianuclear bodies (PML-NBs), which are characterized by the presence of the tripartite motif family protein, PML/TRIM19, and their modification often occurs in response to environmental stresses, including oxidative stress, aging and virus infection [13e16]. Although the biological relevance of SUMOylation promiscuously with ubiquitinylation in PML-NBs remains elusive, the existence of RNF4/SNURF, an E3 ubiquitin ligase that promotes ubiquitinylation of SUMO-modified proteins, in PML-NBs, suggests a role for PMLNBs in controlling the stability of SUMOylated proteins with ubiquitinylation [17e20]. We have previously assessed the distribution of a derivative of puromycin, O-propargyl-puromycin (OP-Puro), in HeLa cells using
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Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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Fig. 1. Puromycin induces SUMO-2/3-positive nuclear foci formation upon proteasome inhibition. (A) Exponentially growing HeLa cells were exposed to vehicle alone (e, upper left), 10 mM puromycin (lower left), 10 mM MG132 (upper right) or 10 mM puromycin plus 10 mM MG132 (lower-right) for 4 h at 37 C, and were then subjected to immunostaining using anti-SUMO-2/3 antibody. Scale bar indicates 20 mm. (B) HeLa cells were cultured and treated as indicated in (A). The proportion of cells forming SUMO-positive nuclear foci was evaluated for each treatment. Values shown represent the mean ± SD of three independent experiments. (C) Exponentially growing HeLa cells were exposed to vehicle alone (e, left column), 10 mM puromycin (Puro, middle left column), 10 mM cycloheximide (CHX, middle column), 10 mM emetine (middle right column) or 10 mM anisomycin (Aniso, right column) in the absence (upper panels) or presence of 10 mM MG132 (lower panels) for 4 h at 37 C, and were then subjected to immunostaining using anti-SUMO-2/3 antibody. Scale bar indicates 20 mm.
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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Fig. 2. Puromycin induces formation of the ubiquitin-positive foci in the cytoplasm and nucleus upon proteasome inhibition. (A) Exponentially growing HeLa cells were exposed to vehicle alone (e, upper left), 10 mM puromycin (lower left), 10 mM MG132 (upper right) or 10 mM puromycin plus 10 mM MG132 (lower right) for 4 h at 37 C, and were then subjected to immunostaining using anti-multi-/poly-ubiquitin (FK2) antibody. Scale bar indicates 20 mm. (B) HeLa cells were exposed to 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C, and were then subjected to immunostaining using anti-SUMO-2/3- (SUMO-2/3, upper left) and FK2 (Ubi, upper right) antibodies. DNA was stained with DAPI (DAPI, upper middle). The merged images of SUMO-2/3 and DAPI staining (lower left), and ubiquitin (FK2) and DAPI staining (lower right) were indicated in the zoomed-in images.
click chemistry and found that substantial OP-Puro signals were detected in nuclear foci containing SUMO-2/3 and ubiquitin when the proteasome inhibitor MG132 was co-administrated [21]. Intriguingly, such OP-Puro-SUMO-2/3-ubiquitin-positive nuclear foci merged with PML-staining, suggesting that OP-Puro accumulated concurrently with SUMO-2/3 and ubiquitin in PML-NBs during impaired proteasome activity. Considering that OP-Puro signals represented OP-puromycylated polypeptides [4,22], these data suggest a role for PML-NBs together with SUMO-2/3 and ubiquitin in nuclear sequestration of puromycylated immature/aberrant polypeptides [21].
In this study, we tested the effect of puromycin, instead of OPPuro, on the subcellular localization of SUMO-2/3 and ubiquitin. When human cervical cancer HeLa cells were exposed to puromycin and MG132, SUMO-2/3 were abundantly detected with ubiquitin in PML-NBs, where RNF4 was also co-localized. Additionally, we revealed the pharmacological conditions for disassembly of the nuclear bodies containing SUMO-2/3 and ubiquitin. Our results indicated that puromycin exposure mimicked the OPPuro effect and provided new insight into the role of the ubiquitin family in nuclear protein homeostasis (nucleo-proteostasis) and intracellular sequestration of immature/aberrant proteins.
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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Fig. 3. PML and RNF4 co-localize with SUMO-2/3-positive nuclear foci in cells co-exposed to puromycin and MG132. (A) HeLa cells treated with 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C were double-stained with anti-SUMO-2/3 (left) and anti-PML (middle) antibodies. Merged image are shown on the right. Scale bar indicates 20 mm. (B) HeLa cells exposed to 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C were double-stained with anti-SUMO-2/3 (left) and anti-RNF4 (middle) antibodies. Merged image are represented on the right. Scale bar indicates 20 mm.
2. Materials and methods
2.3. Statistical analysis
2.1. Cell culture and drug treatments
Unless otherwise stated, all data are presented as the mean ± standard deviation (SD). Within individual experiments, data points were based on a minimum of triplicate representative samples and experiments were repeated at least three times.
HeLa cells were maintained in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 Ham with 5% fetal calf serum and antibiotics at 37 C in 5% CO2 incubator. Puromycin, cycloheximide, emetine and anisomycin were obtained from Wako Wako Pure Chemical Industries. Benzyloxycarbonyl-L-leucyl-L-leucyl-Lleucinal (MG132) was purchased from Peptide Institute Inc. Drugs were dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium. Incubation period was indicated in the text. It should be noted that, during our initial study, we used SCADS Inhibitor Kit provided by Screening Committee of Anticancer Drugs (Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan).
2.2. Antibodies and indirect-immunofluorescence analysis Anti-SUMO-2/3 and anti-SUMO-1 rabbit monoclonal antibodies were obtained from Cell Signaling Technologies. Anti-multi-/polyubiquitin mouse monoclonal (FK2) and anti-PML antibodies were obtained from Medical and Biological Laboratories (MBL) and Santa Cruz Biotechnology, respectively. Anti-human RNF4 mouse monoclonal antibody was a kind gift from Dr. Urano, Shimane University [23,24]. Secondary antibodies used were purchased from Jackson ImmunoResearch, Life technologies and MBL. Indirectimmunofluorescence was previously described [21]. Briefly, cultured cells grown on glass coverslips were fixed with 4% paraformaldehyde, followed by permeabilization with 0/2% TritonX100. Cells were incubated with primary antibody for 1 h, followed by an appropriate secondary antibody. The specimens were analyzed with a DP72 microscope (Olympus). DNA was visualized with 40 , 6-diamidino-2-phenylindole (DAPI).
3. Results and discussion 3.1. Detection of SUMO-positive nuclear foci in puromycin-exposed HeLa cells upon proteasome inhibition To elucidate whether puromycin induces redistribution of SUMO-2/3 in cultured HeLa cells upon proteasome inhibition, we exposed cells to the drug for 4 h in the absence or presence of MG132, followed by indirect immunofluorescence analysis using an anti-SUMO-2/3 antibody. Multiple SUMO-2/3-positive nuclear foci were detected in cells co-administrated with puromycin and MG132, but not in cells exposed to either of the drugs alone (Fig. 1A). In more than 97% of the cells co-exposed to puromycin and MG132, we detected 5e10 SUMO-2/3-positive nuclear foci (Fig. 1B and Supplementary Fig. 1). In addition to SUMO-2/3, SUMO1 appeared to be redistributed to the nuclear foci when cells were co-exposed to puromycin and MG132 (Supplementary Fig. 2), indicating that puromycin induced redistribution of all SUMO proteins (SUMO-2/3 and SUMO-1 subfamilies) to nuclear foci upon proteasome inhibition. Because puromycin exhibited SUMO-2/3positive nuclear foci while other ribosome-binding inhibitors tested so far, including cycloheximide, emetine and anisomycin, hardly induced nuclear foci (Fig. 1C), SUMO redistribution may be due to accumulation of puromycylated immature polypeptides, rather than protein synthesis inhibition per se (Supplementary Fig. 3).
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proteasome activity. Besides HeLa cells, we investigated human bone osteosarcoma U2-OS cells and found formation of SUMO-2/3ubiquitin positive nuclear foci in response to co-administration of puromycin and MG132 (Supplementary Fig. 5), suggesting that the nuclear SUMO-ubiquitin foci assembly by puromycin upon proteasome inhibition was a conserved phenomenon among at least two different human cell lines. It should be noted that ubiquitin foci detected in the cytoplasm did not merge with the SUMO-2/3staining (Fig. 2B), indicating that SUMO-2/3 was not a major component of the cytoplasmic foci. 3.3. PML and RNF4 co-localize with SUMO-2/3-positive nuclear foci
Fig. 4. Assembly and disassembly of SUMO-2/3-ubiquitin-positive nuclear foci are pharmacologically manipulable. (A) Exponentially growing HeLa cells were exposed to 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C. After washing cells with drugfree medium, cells were cultured without puromycin and MG132 for 0, 4 and 8 h at 37 C, and were then subjected to double-staining with anti-SUMO-2/3 and antiubiquitin (FK2) antibodies. The ratio of cells representing SUMO-2/3-ubiquitin (FK2)positive nuclear foci was evaluated for each treatment. The value shown represent the mean ± SD of three independent experiments. (B) The model illustrates the involvement of SUMO and ubiquitin in PML-NB assembly/disassembly and the possible points of action of puromycin and MG132.
3.2. Redistribution of ubiquitin occurs abundantly with SUMO-2/3 redistribution to the nuclear foci We next investigated ubiquitin redistribution in response to coadministration of puromycin and MG132. HeLa cells were exposed to puromycin for 4 h in the absence or presence of MG132, followed by indirect immunofluorescence analysis using an anti-multi-/ poly-ubiquitin (FK2) antibody. We found that multiple ubiquitinpositive foci were formed in the cytoplasm upon exposure to puromycin alone, whereas intense ubiquitin-positive foci formation both in the cytoplasm and nucleus was induced in cells co-exposed to puromycin and MG132 (Fig. 2A, B and Supplementary Fig. 4), indicating that ubiquitin assembly occurred concurrently with SUMO-2/3 assembly in response to puromycin during impaired
Because SUMO-2/3 and ubiquitin play important roles in PMLNB formation [13e16], we assumed that SUMO-2/3-ubiquitinpositive nuclear foci upon co-administration of puromycin and MG132 may represent assembly of PML-NBs. To assess this hypothesis, antibodies against two of the well-recognized PML-NB components, PML and RNF4 [17e20], were used in indirect immunofluorescence analysis. As shown in Fig. 3A, B and Supplementary Fig. 6, both anti-PML and anti-RNF4 antibodies stained the SUMO-2/3-positive nuclear foci, indicating that the SUMO-2/3-positive nuclear foci represented PML-NBs, implying that assembly of PML-NBs was substantially enhanced by coadministration of puromycin and MG132. Given that RNF4 is a SUMO-targeted E3 ubiquitin ligase, we predicted that puromycin-MG132-induced RNF4 recruitment to PML-NBs may precede ubiquitin accumulation in PML-NBs. To test this scenario, we treated cells with RNF4 siRNA and examined whether puromycin induced ubiquitin assembly in the nucleus upon proteasome inhibition. Unfortunately, we detected a negligible decrease in appearance of ubiquitin-positive nuclear foci in cells treated with RNF4 siRNA compared with that of control siRNAtreated cells (Supplementary Fig. 7A and B). These results suggest that ubiquitin assembly in PML-NBs was independent of RNF4 and/ or imply the existence of SUMO-targeted E3 ubiquitin ligase(s), besides RNF4, which may be activated in response to puromycin exposure during impaired proteasome activity. Alternatively, ubiquitin was recruited to PML-NBs via a mechanism fully independent of SUMO-targeted ubiquitin ligase activity. The molecular regulation of ubiquitin assembly in PML-NBs in cells co-exposed to puromycin and MG132 requires further studies in the future. 3.4. SUMO-2/3-ubiquitin nuclear foci are disassembled in cells under drug-free conditions To elucidate whether assembly and disassembly of the SUMO-2/ 3-ubiquitin-positive nuclear foci are reversibly regulated, puromycin-MG132-treated HeLa cells were incubated in a drug-free medium for up to 8 h at 37 C, and then subjected to immunostaining using anti-SUMO-2/3 and anti-ubiquitin (FK2) antibodies (Supplementary Fig. 8). As summarized in Fig. 4A, the proportion of cells containing SUMO-2/3-ubiquitin nuclear foci gradually decreased by 2 h after the medium change, and after 4e8 h of incubation in the drug-free medium, there were almost no cells containing SUMO-2/3-ubiquitin nuclear foci. These results indicated that signal(s) that enhanced assembly and/or inhibited disassembly of SUMOs and ubiquitin in PML-NBs were attenuated upon removal of the drugs, implying that formation of the SUMOubiquitin nuclear foci was a reversible and pharmacologically manipulable process. Finally, based on our observations, we propose a model that predicts the mechanism by which the puromycin-MG132-induced signal(s) controls the assembly and/or disassembly of PML-NBs via the SUMO-ubiquitin pathway (Fig. 4B). While key questions
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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concerning the target(s) of SUMOylation and ubiquitinylation at PML-NBs in response to puromycin during impaired proteasome activity remain to be clarified, we believe that the pharmacological conditions presented here will be beneficial for researchers who are interested in investigating the regulation of PML-assembly/ disassembly via the SUMO and ubiquitin pathways. Understanding this regulation at the molecular level will not only help elucidate the not-yet-well established idea of the protective cellular response involving nuclear sequestration of aberrant proteins [21], but will also contribute to the development of molecular strategies for elimination of toxic aggregated protein species involved in numerous neurodegenerative diseases and other disorders [25,26]. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements We thank all the members of the Saitoh Laboratory for helpful discussion. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant 26116517 (Grant-in-Aid for Scientific Research on Innovative Areas). We also thank Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan, for providing us SCADS Inhibitor Kit. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.05.025. References [1] S. Pestka, Inhibitors of ribosome functions, Annu. Rev. Microbiol. 25 (1971) 487e562. [2] D. Nathans, Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains, Proc. Natl. Acad. Sci. U. S. A. 51 (1964) 585e592. [3] E.K. Schmidt, G. Clavarino, M. Ceppi, P. Pierre, SUnSET, a nonradioactive method to monitor protein synthesis, Nat. Methods 6 (2009) 275e277. [4] J. Liu, Y. Xu, D. Stoleru, A. Salic, Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 413e418. [5] A. David, B.P. Dolan, H.D. Hickman, J.J. Knowlton, G. Clavarino, P. Pierre, J.R. Bennink, J.W. Yewdell, Nuclear translation visualized by ribosome-bound nascent chain puromycylation, J. Cell Biol. 197 (2012) 45e57. [6] F. Wang, L.A. Durfee, J.M. Huibregtse, A cotranslational ubiquitination pathway for quality control of misfolded proteins, Mol. Cell 50 (2013)
368e378. [7] H. Saitoh, J. Hinchey, Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3, J. Biol. Chem. 275 (2000) 6252e6258. [8] A. Floth, F. Melchior, Sumoylation: a regulatory protein modification in health and disease, Annu. Rev. Biochem. 82 (2013) 357e385. [9] F. Golebiowski, I. Matic, M.H. Tatham, C. Cole, Y. Yin, A. Nakamura, J. Cox, G.J. Barton, M. Mann, R.T. Hay, System-wide changes to SUMO modifications in response to heat shock, Sci. Signal. 2 (2009) ra24. , D. Brezinov da, J. Lipov, T. Ruml, Z. Knejzlík, SUMO-2/3 [10] M. Castor alova a, M. Sve conjugates accumulating under heat shock or MG132 treatment result largely from new protein synthesis, Biochim. Biophys. Acta 1823 (2012) 911e919. [11] G.J. Praefcke, K. Hofmann, R.J. Dohmen, SUMO playing tag with ubiquitin, Trends Biochem. Sci. 37 (2012) 23e31. [12] M. Nie, M.N. Boddy, Cooperativity of the SUMO and ubiquitin pathways in genome stability, Biomolecules 6 (2016) pii: E14. [13] R. Bernardi, P.P. Pandolfi, Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies, Nat. Rev. Mol. Cell Biol. 8 (2007) 1006e1016. [14] C. Boutell, R.D. Everett, Regulation of alphaherpesvirus infections by the ICP0 family of proteins, J. Gen. Virol. 94 (2013) 465e481. [15] X. Cheng, H.Y. Kao, Post-translational modifications of PML: consequences and implications, Front. Oncol. 2 (2013) 210. , V. Lallemand-Breitenbach, PML nuclear bodies: assembly [16] U. Sahin, H. de The and oxidative stress-sensitive sumoylation, Nucleus 5 (2014) 499e507. [17] M. H€ akli, U. Karvonen, O.A. J€ anne, J.J. Palvimo, SUMO-1 promotes association of SNURF (RNF4) with PML nuclear bodies, Exp. Cell Res. 304 (2005) 224e233. [18] M.H. Tatham, M.C. Geoffroy, L. Shen, A. Plechanovova, N. Hattersley, E.G. Jaffray, J.J. Palvimo, R.T. Hay, RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation, Nat. Cell Biol. 10 (2008) 538e546. [19] V. Lallemand-Breitenbach, M. Jeanne, S. Benhenda, R. Nasr, M. Lei, L. Peres, , Arsenic degrades PML or PML-RARalpha J. Zhou, J. Zhu, B. Raught, H. de The through a SUMO-triggered RNF4/ubiquitin-mediated pathway, Nat. Cell Biol. 10 (2008) 547e555. [20] L. Guo, B.I. Giasson, A. Glavis-Bloom, M.D. Brewer, J. Shorter, A.D. Gitler, X. Yang, A cellular system that degrades misfolded proteins and protects against neurodegeneration, Mol. Cell 55 (2014) 15e30. [21] N. Uozumi, H. Matsumoto, H. Saitoh, Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities, Biochem. Biophys. Res. Commun. 474 (2016) 247e251. [22] R.A. Signer, J.A. Magee, A. Salic, S.J. Morrison, Haematopoietic stem cells require a highly regulated protein synthesis rate, Nature 509 (2014) 49e54. [23] M. Saito, Y. Fujimitsu, T. Sasano, Y. Yoshikai, R. Ban-Ishihara, Y. Nariai, T. Urano, H. Saitoh, The SUMO-targeted ubiquitin ligase RNF4 localizes to etoposide-exposed mitotic chromosomes: implication for a novel DNA damage response during mitosis, Biochem. Biophys. Res. Commun. 447 (2014) 83e138. [24] T. Moriyama, Y. Fujimitsu, Y. Yoshikai, T. Sasano, K. Yamada, M. Murakami, T. Urano, K. Sugasawa, H. Saitoh, SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells, Biochem. Biophys. Res. Commun. 447 (2014) 419e424. [25] E.T. Powers, R.I. Morimoto, A. Dillin, J.W. Kelly, W.E. Balch, Biological and chemical approaches to diseases of proteostasis deficiency, Annu. Rev. Biochem. 78 (2009) 959e991. [26] T.P. Knowles, M. Vendruscolo, C.M. Dobson, The amyloid state and its association with protein misfolding diseases, Nat. Rev. Mol. Cell Biol. 15 (2014) 384e396.
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition Hotaru Matsumoto a, Hisato Saitoh a, b, * a b
Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto, Japan Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2016 Accepted 3 May 2016 Available online xxx
We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. © 2016 Elsevier Inc. All rights reserved.
Keywords: Nuclear protein sequestration Puromycin Small ubiquitin-related modifier (SUMO) Ubiquitin proteasome system Promyelocytic leukemia nuclear bodies
1. Introduction Puromycin is an aminonucleoside antibiotic produced by Streptomyces alboniger [1]. When administrated to cells, it enters the ribosome A site, followed by incorporation into the nascent polypeptide chain, elongating either on the monoribosome or polyribosomes, which leads to covalent attachment of puromycin to the C-terminal end of the nascent polypeptides, referred to as ‘puromycylation’ [2e5]. Because puromycylation prematurely terminates elongation at any given site in the elongating polypeptide, the resulting puromycylated polypeptides are expected to be heterogeneous in size and improperly folded. Under standard conditions, such puromycylated polypeptides are subjected to an elimination reaction controlled by the ubiquitin-proteasome system (UPS) [6]. However, when the UPS is perturbed in cells, puromycylated immature polypeptides are expected to accumulate,
Abbreviations: SUMO, small ubiquitin-related modifier; PML-NBs, promyelocytic leukemia-nuclear bodies; RNF4, really interesting new gene (RING) finger protein 4; DAPI, 40 , 6-diamidino-2-phenylindole; OP-Puro, O-propargyl-puromycin. * Corresponding author. Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto, 860-8555, Japan. E-mail address: [email protected] (H. Saitoh).
causing cytotoxic effects, including stress response and growth defects, ultimately inducing cell death. To escape such a scenario, cells should contain an intrinsic protection system that would respond to accumulation of immature/aberrant proteins. However, the cellular defense strategy against immature/aberrant polypeptides remains largely unknown. The small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are highly conserved proteins, which can be covalently attached to multiple cellular proteins, referred to as SUMOylation [7,8]. Intriguingly, SUMOylation frequently, but not always, occurs with ubiquitin modification (ubiquitinylation) [9e12]. Many SUMOs and SUMOyalted proteins are detected in promyelocytic leukemianuclear bodies (PML-NBs), which are characterized by the presence of the tripartite motif family protein, PML/TRIM19, and their modification often occurs in response to environmental stresses, including oxidative stress, aging and virus infection [13e16]. Although the biological relevance of SUMOylation promiscuously with ubiquitinylation in PML-NBs remains elusive, the existence of RNF4/SNURF, an E3 ubiquitin ligase that promotes ubiquitinylation of SUMO-modified proteins, in PML-NBs, suggests a role for PMLNBs in controlling the stability of SUMOylated proteins with ubiquitinylation [17e20]. We have previously assessed the distribution of a derivative of puromycin, O-propargyl-puromycin (OP-Puro), in HeLa cells using
http://dx.doi.org/10.1016/j.bbrc.2016.05.025 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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Fig. 1. Puromycin induces SUMO-2/3-positive nuclear foci formation upon proteasome inhibition. (A) Exponentially growing HeLa cells were exposed to vehicle alone (e, upper left), 10 mM puromycin (lower left), 10 mM MG132 (upper right) or 10 mM puromycin plus 10 mM MG132 (lower-right) for 4 h at 37 C, and were then subjected to immunostaining using anti-SUMO-2/3 antibody. Scale bar indicates 20 mm. (B) HeLa cells were cultured and treated as indicated in (A). The proportion of cells forming SUMO-positive nuclear foci was evaluated for each treatment. Values shown represent the mean ± SD of three independent experiments. (C) Exponentially growing HeLa cells were exposed to vehicle alone (e, left column), 10 mM puromycin (Puro, middle left column), 10 mM cycloheximide (CHX, middle column), 10 mM emetine (middle right column) or 10 mM anisomycin (Aniso, right column) in the absence (upper panels) or presence of 10 mM MG132 (lower panels) for 4 h at 37 C, and were then subjected to immunostaining using anti-SUMO-2/3 antibody. Scale bar indicates 20 mm.
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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Fig. 2. Puromycin induces formation of the ubiquitin-positive foci in the cytoplasm and nucleus upon proteasome inhibition. (A) Exponentially growing HeLa cells were exposed to vehicle alone (e, upper left), 10 mM puromycin (lower left), 10 mM MG132 (upper right) or 10 mM puromycin plus 10 mM MG132 (lower right) for 4 h at 37 C, and were then subjected to immunostaining using anti-multi-/poly-ubiquitin (FK2) antibody. Scale bar indicates 20 mm. (B) HeLa cells were exposed to 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C, and were then subjected to immunostaining using anti-SUMO-2/3- (SUMO-2/3, upper left) and FK2 (Ubi, upper right) antibodies. DNA was stained with DAPI (DAPI, upper middle). The merged images of SUMO-2/3 and DAPI staining (lower left), and ubiquitin (FK2) and DAPI staining (lower right) were indicated in the zoomed-in images.
click chemistry and found that substantial OP-Puro signals were detected in nuclear foci containing SUMO-2/3 and ubiquitin when the proteasome inhibitor MG132 was co-administrated [21]. Intriguingly, such OP-Puro-SUMO-2/3-ubiquitin-positive nuclear foci merged with PML-staining, suggesting that OP-Puro accumulated concurrently with SUMO-2/3 and ubiquitin in PML-NBs during impaired proteasome activity. Considering that OP-Puro signals represented OP-puromycylated polypeptides [4,22], these data suggest a role for PML-NBs together with SUMO-2/3 and ubiquitin in nuclear sequestration of puromycylated immature/aberrant polypeptides [21].
In this study, we tested the effect of puromycin, instead of OPPuro, on the subcellular localization of SUMO-2/3 and ubiquitin. When human cervical cancer HeLa cells were exposed to puromycin and MG132, SUMO-2/3 were abundantly detected with ubiquitin in PML-NBs, where RNF4 was also co-localized. Additionally, we revealed the pharmacological conditions for disassembly of the nuclear bodies containing SUMO-2/3 and ubiquitin. Our results indicated that puromycin exposure mimicked the OPPuro effect and provided new insight into the role of the ubiquitin family in nuclear protein homeostasis (nucleo-proteostasis) and intracellular sequestration of immature/aberrant proteins.
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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Fig. 3. PML and RNF4 co-localize with SUMO-2/3-positive nuclear foci in cells co-exposed to puromycin and MG132. (A) HeLa cells treated with 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C were double-stained with anti-SUMO-2/3 (left) and anti-PML (middle) antibodies. Merged image are shown on the right. Scale bar indicates 20 mm. (B) HeLa cells exposed to 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C were double-stained with anti-SUMO-2/3 (left) and anti-RNF4 (middle) antibodies. Merged image are represented on the right. Scale bar indicates 20 mm.
2. Materials and methods
2.3. Statistical analysis
2.1. Cell culture and drug treatments
Unless otherwise stated, all data are presented as the mean ± standard deviation (SD). Within individual experiments, data points were based on a minimum of triplicate representative samples and experiments were repeated at least three times.
HeLa cells were maintained in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 Ham with 5% fetal calf serum and antibiotics at 37 C in 5% CO2 incubator. Puromycin, cycloheximide, emetine and anisomycin were obtained from Wako Wako Pure Chemical Industries. Benzyloxycarbonyl-L-leucyl-L-leucyl-Lleucinal (MG132) was purchased from Peptide Institute Inc. Drugs were dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium. Incubation period was indicated in the text. It should be noted that, during our initial study, we used SCADS Inhibitor Kit provided by Screening Committee of Anticancer Drugs (Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan).
2.2. Antibodies and indirect-immunofluorescence analysis Anti-SUMO-2/3 and anti-SUMO-1 rabbit monoclonal antibodies were obtained from Cell Signaling Technologies. Anti-multi-/polyubiquitin mouse monoclonal (FK2) and anti-PML antibodies were obtained from Medical and Biological Laboratories (MBL) and Santa Cruz Biotechnology, respectively. Anti-human RNF4 mouse monoclonal antibody was a kind gift from Dr. Urano, Shimane University [23,24]. Secondary antibodies used were purchased from Jackson ImmunoResearch, Life technologies and MBL. Indirectimmunofluorescence was previously described [21]. Briefly, cultured cells grown on glass coverslips were fixed with 4% paraformaldehyde, followed by permeabilization with 0/2% TritonX100. Cells were incubated with primary antibody for 1 h, followed by an appropriate secondary antibody. The specimens were analyzed with a DP72 microscope (Olympus). DNA was visualized with 40 , 6-diamidino-2-phenylindole (DAPI).
3. Results and discussion 3.1. Detection of SUMO-positive nuclear foci in puromycin-exposed HeLa cells upon proteasome inhibition To elucidate whether puromycin induces redistribution of SUMO-2/3 in cultured HeLa cells upon proteasome inhibition, we exposed cells to the drug for 4 h in the absence or presence of MG132, followed by indirect immunofluorescence analysis using an anti-SUMO-2/3 antibody. Multiple SUMO-2/3-positive nuclear foci were detected in cells co-administrated with puromycin and MG132, but not in cells exposed to either of the drugs alone (Fig. 1A). In more than 97% of the cells co-exposed to puromycin and MG132, we detected 5e10 SUMO-2/3-positive nuclear foci (Fig. 1B and Supplementary Fig. 1). In addition to SUMO-2/3, SUMO1 appeared to be redistributed to the nuclear foci when cells were co-exposed to puromycin and MG132 (Supplementary Fig. 2), indicating that puromycin induced redistribution of all SUMO proteins (SUMO-2/3 and SUMO-1 subfamilies) to nuclear foci upon proteasome inhibition. Because puromycin exhibited SUMO-2/3positive nuclear foci while other ribosome-binding inhibitors tested so far, including cycloheximide, emetine and anisomycin, hardly induced nuclear foci (Fig. 1C), SUMO redistribution may be due to accumulation of puromycylated immature polypeptides, rather than protein synthesis inhibition per se (Supplementary Fig. 3).
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proteasome activity. Besides HeLa cells, we investigated human bone osteosarcoma U2-OS cells and found formation of SUMO-2/3ubiquitin positive nuclear foci in response to co-administration of puromycin and MG132 (Supplementary Fig. 5), suggesting that the nuclear SUMO-ubiquitin foci assembly by puromycin upon proteasome inhibition was a conserved phenomenon among at least two different human cell lines. It should be noted that ubiquitin foci detected in the cytoplasm did not merge with the SUMO-2/3staining (Fig. 2B), indicating that SUMO-2/3 was not a major component of the cytoplasmic foci. 3.3. PML and RNF4 co-localize with SUMO-2/3-positive nuclear foci
Fig. 4. Assembly and disassembly of SUMO-2/3-ubiquitin-positive nuclear foci are pharmacologically manipulable. (A) Exponentially growing HeLa cells were exposed to 10 mM puromycin plus 10 mM MG132 for 4 h at 37 C. After washing cells with drugfree medium, cells were cultured without puromycin and MG132 for 0, 4 and 8 h at 37 C, and were then subjected to double-staining with anti-SUMO-2/3 and antiubiquitin (FK2) antibodies. The ratio of cells representing SUMO-2/3-ubiquitin (FK2)positive nuclear foci was evaluated for each treatment. The value shown represent the mean ± SD of three independent experiments. (B) The model illustrates the involvement of SUMO and ubiquitin in PML-NB assembly/disassembly and the possible points of action of puromycin and MG132.
3.2. Redistribution of ubiquitin occurs abundantly with SUMO-2/3 redistribution to the nuclear foci We next investigated ubiquitin redistribution in response to coadministration of puromycin and MG132. HeLa cells were exposed to puromycin for 4 h in the absence or presence of MG132, followed by indirect immunofluorescence analysis using an anti-multi-/ poly-ubiquitin (FK2) antibody. We found that multiple ubiquitinpositive foci were formed in the cytoplasm upon exposure to puromycin alone, whereas intense ubiquitin-positive foci formation both in the cytoplasm and nucleus was induced in cells co-exposed to puromycin and MG132 (Fig. 2A, B and Supplementary Fig. 4), indicating that ubiquitin assembly occurred concurrently with SUMO-2/3 assembly in response to puromycin during impaired
Because SUMO-2/3 and ubiquitin play important roles in PMLNB formation [13e16], we assumed that SUMO-2/3-ubiquitinpositive nuclear foci upon co-administration of puromycin and MG132 may represent assembly of PML-NBs. To assess this hypothesis, antibodies against two of the well-recognized PML-NB components, PML and RNF4 [17e20], were used in indirect immunofluorescence analysis. As shown in Fig. 3A, B and Supplementary Fig. 6, both anti-PML and anti-RNF4 antibodies stained the SUMO-2/3-positive nuclear foci, indicating that the SUMO-2/3-positive nuclear foci represented PML-NBs, implying that assembly of PML-NBs was substantially enhanced by coadministration of puromycin and MG132. Given that RNF4 is a SUMO-targeted E3 ubiquitin ligase, we predicted that puromycin-MG132-induced RNF4 recruitment to PML-NBs may precede ubiquitin accumulation in PML-NBs. To test this scenario, we treated cells with RNF4 siRNA and examined whether puromycin induced ubiquitin assembly in the nucleus upon proteasome inhibition. Unfortunately, we detected a negligible decrease in appearance of ubiquitin-positive nuclear foci in cells treated with RNF4 siRNA compared with that of control siRNAtreated cells (Supplementary Fig. 7A and B). These results suggest that ubiquitin assembly in PML-NBs was independent of RNF4 and/ or imply the existence of SUMO-targeted E3 ubiquitin ligase(s), besides RNF4, which may be activated in response to puromycin exposure during impaired proteasome activity. Alternatively, ubiquitin was recruited to PML-NBs via a mechanism fully independent of SUMO-targeted ubiquitin ligase activity. The molecular regulation of ubiquitin assembly in PML-NBs in cells co-exposed to puromycin and MG132 requires further studies in the future. 3.4. SUMO-2/3-ubiquitin nuclear foci are disassembled in cells under drug-free conditions To elucidate whether assembly and disassembly of the SUMO-2/ 3-ubiquitin-positive nuclear foci are reversibly regulated, puromycin-MG132-treated HeLa cells were incubated in a drug-free medium for up to 8 h at 37 C, and then subjected to immunostaining using anti-SUMO-2/3 and anti-ubiquitin (FK2) antibodies (Supplementary Fig. 8). As summarized in Fig. 4A, the proportion of cells containing SUMO-2/3-ubiquitin nuclear foci gradually decreased by 2 h after the medium change, and after 4e8 h of incubation in the drug-free medium, there were almost no cells containing SUMO-2/3-ubiquitin nuclear foci. These results indicated that signal(s) that enhanced assembly and/or inhibited disassembly of SUMOs and ubiquitin in PML-NBs were attenuated upon removal of the drugs, implying that formation of the SUMOubiquitin nuclear foci was a reversible and pharmacologically manipulable process. Finally, based on our observations, we propose a model that predicts the mechanism by which the puromycin-MG132-induced signal(s) controls the assembly and/or disassembly of PML-NBs via the SUMO-ubiquitin pathway (Fig. 4B). While key questions
Please cite this article in press as: H. Matsumoto, H. Saitoh, Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.025
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concerning the target(s) of SUMOylation and ubiquitinylation at PML-NBs in response to puromycin during impaired proteasome activity remain to be clarified, we believe that the pharmacological conditions presented here will be beneficial for researchers who are interested in investigating the regulation of PML-assembly/ disassembly via the SUMO and ubiquitin pathways. Understanding this regulation at the molecular level will not only help elucidate the not-yet-well established idea of the protective cellular response involving nuclear sequestration of aberrant proteins [21], but will also contribute to the development of molecular strategies for elimination of toxic aggregated protein species involved in numerous neurodegenerative diseases and other disorders [25,26]. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements We thank all the members of the Saitoh Laboratory for helpful discussion. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant 26116517 (Grant-in-Aid for Scientific Research on Innovative Areas). We also thank Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from The Ministry of Education, Culture, Sports, Science and Technology, Japan, for providing us SCADS Inhibitor Kit. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.05.025. References [1] S. Pestka, Inhibitors of ribosome functions, Annu. Rev. Microbiol. 25 (1971) 487e562. [2] D. Nathans, Puromycin inhibition of protein synthesis: incorporation of puromycin into peptide chains, Proc. Natl. Acad. Sci. U. S. A. 51 (1964) 585e592. [3] E.K. Schmidt, G. Clavarino, M. Ceppi, P. Pierre, SUnSET, a nonradioactive method to monitor protein synthesis, Nat. Methods 6 (2009) 275e277. [4] J. Liu, Y. Xu, D. Stoleru, A. Salic, Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 413e418. [5] A. David, B.P. Dolan, H.D. Hickman, J.J. Knowlton, G. Clavarino, P. Pierre, J.R. Bennink, J.W. Yewdell, Nuclear translation visualized by ribosome-bound nascent chain puromycylation, J. Cell Biol. 197 (2012) 45e57. [6] F. Wang, L.A. Durfee, J.M. Huibregtse, A cotranslational ubiquitination pathway for quality control of misfolded proteins, Mol. Cell 50 (2013)
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