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Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities
Biochemical and Biophysical Research Communications 474 (2016) 247e251
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities Naoki Uozumi a, 1, Hotaru Matsumoto a, 1, 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 23 March 2016 Accepted 30 March 2016 Available online 25 April 2016
The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycinconjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis. © 2016 Elsevier Inc. All rights reserved.
Keywords: Protein homeostasis Puromycin Click chemistry Small ubiquitin-related modifier (SUMO) Ubiquitin Promyelocytic leukemia nuclear bodies
1. Introduction Puromycin, an amino-nucleoside antibiotic produced by Streptomyces alboniger, is a potent protein synthesis inhibitor in both prokaryotic and eukaryotic cells [1]. Puromycin enters the ribosome A site and terminates translation by ribosome-catalyzed covalent incorporation into the nascent polypeptide chain, referred to as ‘puromycylation’ [2e8]. Because this generates prematurely terminated and misfolded polypeptides, puromycin-conjugated (puromycylated) polypeptides are likely to cause cytotoxic effects and thus may contribute to induction of the stress response and growth defects; however, regulation of their trafficking and fate in vital cellular systems remain largely uncharacterized. Promyelocytic leukemia-nuclear bodies (PML-NBs), also known
Abbreviations: OP-Puro, O-propargyl-puromycin; SUMO, small ubiquitin-related modifier; PML-NBs, promyelocytic leukemia-nuclear bodies; DAPI, 40 ,6-diamidino2-phenylindole. * 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). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.03.155 0006-291X/© 2016 Elsevier Inc. All rights reserved.
as nuclear dot 10 (ND10) or PML oncogenic domains (PODs), are nuclear domains characterized by the presence of the tripartite motif family protein PML/TRIM19 [9e13]. PML-NBs are involved in a wide variety of cellular activities, including the stress response, aging and viral infection [9e13]. A wide variety of proteins are assembled into PML-NBs via post-translational modification of PML and/or its interactors mediated by the SUMO-2/3 and ubiquitin conjugation pathways. This may contribute to a protective response involving the intra-nuclear sequestration, storage or degradation of non-functional and aberrant proteins [9e13]. Here we use a puromycin derivative, O-propargyl-puromycin (OP-Puro) to monitor puromycylated polypeptides in cultured HeLa cells. OP-Puro is incorporated into elongating polypeptides on ribosomes in a manner similar to puromycin and can be fluorescently labeled by an azide-alkyne reaction, called ‘click chemistry’ [4e8]. This enables the subcellular distribution of OP-puromycylated polypeptides to be visualized in cells. Notably, PO-Puro signals are detected in the cytoplasm and nucleus with substantial concentration at the nucleolus under standard culture conditions. Intriguingly, OP-Puro signals are detected at multiple small nuclear foci when the proteasome inhibitor MG132 is co-administrated. These small nuclear foci may represent PML-NBs, because PML,
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SUMO-2/3 and ubiquitin co-accumulate at the foci. Our findings suggest a role for the nucleus together with the SUMO and ubiquitin pathways in protein homeostasis and the intracellular sequestration of immature polypeptides.
2. Materials and methods 2.1. Cell culture and drug treatments HeLa cells were cultured 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. Protein synthesis inhibitors, puromycin, cycloheximide, emetin and anisomycin, were obtained from Wako Pure Chemical Industries. O-propargyl-puromycin (OP-Puro) was obtained from Jena Bioscience. Benzyloxycarbonyl-L-leucyl-Lleucyl-L-leucinal (MG132) was purchased from Peptide Institute Inc. Each drug was dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium as indicated in the text.
2.2. Antibodies Anti-SUMO-2/3 and -SUMO-1 rabbit monoclonal antibodies were obtained from Cell Signaling Technologies. Anti-polyubiquitin mouse monoclonal (FK2), anti-PML and anti-nucleolin antibodies were obtained from Medical and Biological Laboratories (MBL), Santa Cruz Biotechnology and MerckMillipore, respectively. Secondary antibodies used were purchased from Jackson ImmunoResearch, Life technologies and MBL.
2.3. OP-Puro labeling of cultured HeLa cells and detection by fluorescence microscopy OP-Puro labeling was carried out as previously described [5]. In brief, HeLa cells were grown on glass coverslips in culture medium. To visualize OP-Puro incorporation, we typically exposed cells to culture medium containing 10 mM OP-Puro and 10 mM MG132 for 4 h at 37 C. The cells were washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were washed with PBS, followed by permeabilization with PBS containing 0.1% Triton X-100, and then washed with PBS. OP-Puro was detected by performing an azidealkyne cycloaddition for 30 min at room temperature using the Click-iT Cell Reaction Buffer Kit containing 15 mM Alexa 488/555-N3 azide (Life Technologies). After staining, the coverslips were washed several times with PBS and counterstained with 40 ,6diamidino-2-phenylindole (DAPI). If indirect-immunofluorescence was performed using specific antibodies to co-localize OP-Puro signals, the cells were incubated with the appropriate antibody and the indirect-immunofluorescence reaction continued as described below. Fluorescence images were obtained using a DP72 microscope (Olympus).
2.4. Indirect-immunofluorescence analysis Indirect-immunofluorescence was performed as described previously [14,15]. Briefly, HeLa cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were blocked in PBS containing 0.2% bovine
Fig. 1. OP-Puro signals accumulated in the nucleus under standard conditions, and were enhanced during abortive proteasome activities. (A) Schematic representation of the effects of OP-Puro/puromycin. OP-Puro/puromycin, leads to premature release of nascent polypeptides by puromycylation and thus inhibiting polypeptide chain elongation on the ribosome and releasing puromycylated immature polypeptides. In the case of OP-puromycylated polypeptides, their subcellular localization can be visualized by click reaction with fluorescence-conjugated N3. B) Exponentially growing HeLa cells under standard culture conditions were exposed to 10 mM Puro in the absence (upper panels) or presence (bottom panels) of 10 mM MG132 for the indicated periods at 37 C, and were then subjected to the click reaction using Alexa 488-N3 to visualize OP-Puro localization. Bar indicates 20 mm.
N. Uozumi et al. / Biochemical and Biophysical Research Communications 474 (2016) 247e251
serum albumin, 0.1% Triton X-100 for 20 min and incubated with primary antibody for 1 h, followed by an appropriate secondary antibody. The coverslips were analyzed with microscope. DNA was visualized with DAPI.
3. Results and discussion 3.1. Detection of OP-Puro-positive nuclear foci in HeLa cells To elucidate OP-Puro localization in cultured HeLa cells, we exposed cells to OP-Puro for 0, 3, 10, 30, 90 and 240 min, followed by fixation and ‘click chemistry’ (Fig. 1A and B). OP-Puro staining under our experimental conditions for short incubation periods (3e10 min) produced very similar staining patterns to those described previously by several groups [4e8]; signals appeared after incubation for as little as 3 min and the signals were distributed diffusely throughout the cell. Intriguingly, after incubation for 10 min, OP-Puro stained large foci (diameter >1 mm) in the nucleus. In many cells, approximately two to four large OP-Puro-stained foci were detected in nuclei (Fig. 1A, B and Supplementary Fig. 1). We also observed multiple OP-Puro-positive foci in the cytoplasm when cells were exposed to OP-Puro for longer periods (90e240 min; Fig. 1A, B and Supplementary Fig. 1). It should be noted that neither nuclear nor cytoplasmic OP-Puro-positive staining were visible when ribosome-binding translation inhibitors, such as cycloheximide, emetine or anisomycin, were co-
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administrated with OP-Puro (Supplementary Fig. 2), indicating that OP-Puro signals were not due to non-specific products of click chemistry and that the signals represented products associated with ribosome-mediated translation activity. Puromycylated polypeptides are subject to rapid degradation via the ubiquitin-proteasome system (UPS) [16]; therefore, we predicted that OP-Puro signals would be enhanced if proteasome activities were inhibited. As expected, OP-Puro staining was enhanced in cells during abortive proteasome activities. Additionally, we found that multiple small OP-Puro-positive foci (diameter < 1 mm) became visible in the nucleus (Fig. 1A, B and Supplementary Fig. 1), suggesting the existence of nuclear spots where degradation-defective, puromycylated immature polypeptides were accumulated. Taken together, our results indicated that, during longer exposure to OP-Puro and abortive proteasome activities, OP-Puro signals were not randomly distributed throughout the cell; indeed there were multiple regions which facilitated OP-Puro accumulation in the nucleus.
3.2. OP-Puro-positive large and small nuclear foci represented the nucleolus and PML-nuclear bodies, respectively To elucidate the functions of OP-Puro-positive foci in the nucleus, we aimed to identify the components associated with these foci using specific antibodies. To this end, indirectimmunofluorescence using antibodies against nucleolin, a well-
Fig. 2. Nuclear OP-Puro preferentially accumulated in the nucleolus and PML-NBs. (A) Hela cells were exposed to 10 mM Puro in the absence (left column) or presence (right column) of 10 mM MG132 for 240 min at 37 C, and were then subjected to the click reaction using Alexa 488-N3 (upper panels) and immunostaining using an anti-nucleolin antibody (upper-middle panels). Merged images of click reaction and nucleolin-staining are shown in the lower-middle panel. DNA was visualized with DAPI staining (bottom panels). Bar indicates 20 mm. (B) Hela cells were exposed to 10 mM Puro in the absence (left column) or presence (right column) of 10 mM MG132 for 240 min at 37 C, and were then subjected to the click reaction using Alexa 488-N3 (upper panels) and immunostaining using an anti-PML antibody (upper-middle panels). Merged images of click reaction and nucleolin-staining are shown in the lower-middle panel. DNA was visualized with DAPI staining (bottom panels). Bar indicates 20 mm.
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Fig. 3. SUMO-2/3 and ubiquitin were merged with OP-Puro staining at small nuclear foci. (A) Hela cells were exposed to no drug (upper), 10 mM OP-Puro (upper-middle), 10 mM MG132 (lower-middle) or 10 mM OP-Puro plus 10 mM MG132 (lower) for 240 min at 37 C, and were then subjected to the click reaction using Alexa 488-N3 (left column) and immunostaining using an anti-SUMO-2/3 antibody (center column). Merged images are shown at the right. (B) Hela cells were exposed to no drug (upper), 10 mM OP-Puro (uppermiddle), 10 mM MG132 (lower-middle) or 10 mM OP-Puro plus 10 mM MG132 (lower) for 240 min at 37 C, and were then subjected to the click reaction using Alexa 555-N3 (left column) and immunostaining using an anti-FK2 ubiquitin antibody (center column). Merged images are shown at the right. (C) Subcellular OP-Puro distribution detected by click chemistry which indicates a putative trafficking pathway of puromycylated immature polypeptides. OP-Puro signals are distributed in the cytoplasm and nucleoplasm. After longer incubation with OP-Puro, the signals tend to form foci in the cytoplasm and to accumulate at the nucleolus in the nucleoplasm. When proteasome activities are aborted by MG132, OP-Puro signals accumulate at PML-NBs together with SUMO and ubiquitin.
known nucleolus-marker protein [17], revealed co-staining with OP-Puro signals at large nuclear foci (Fig. 2A). Additionally, indirectimmunofluorescence using antibodies against puromyelocytic leukemia (PML), a PML-nuclear body (PML-NB) component [9e11], indicated co-localization of PML with small OP-Puro-positive nuclear foci (Fig. 2B). These results suggested that there were two different-types of nuclear compartment where OP-Puro signals accumulated; the large OP-Puro-positive nuclear foci, which were detected both in the absence and presence of MG132, represented the nucleolus, and the small OP-Puro-positive nuclear foci, which appeared only in the presence of MG132, represented PML-NBs. It should be noted that many, but not all, small OP-Puro-positive
nuclear foci merged with PML-staining (Fig. 2B), indicating the existence of unidentified nuclear bodies that contained puromycylated immature polypeptides. 3.3. OP-Puro, SUMO and ubiquitin were promiscuously accumulated at PML-NBs during abortive proteasome activities The SUMO-2/3 and ubiquitin pathways participate in the regulation of PML function and PML-NB formation; therefore, we suspected involvement of these modification pathways for OP-Puro assembly at PML-NBs during abortive proteasome activities. As shown in Fig. 3, anti-SUMO-2/3 antibody and-ubiquitin (FK2)
N. Uozumi et al. / Biochemical and Biophysical Research Communications 474 (2016) 247e251
antibodies, clearly stained small OP-Puro-positive nuclear foci only in cells exposed to OP-Puro and MG132, whereas neither SUMO-2/3 nor ubiquitin staining was merged with OP-Puro staining in the absence of MG132. These results suggested that promiscuous SUMO and ubiquitin assemblies were induced together with the accumulation of OP-Puro signals at PML-NBs, but not at the nucleolus, during abortive proteasome activities. Of note, we found that the ubiquitin signals merged with cytoplasmic foci, indicating involvement of the ubiquitin pathway in the formation of OP-Puropositive cytoplasmic foci. Taken together, we revealed that there were at least three cellular compartments where OP-Puro tended to accumulate (Fig. 3C); one was larger nuclear domains, representing the nucleolus, which were observed both in the absence and presence of MG132. The second was multiple small nuclear foci, representing PML-NBs, which were detectable during abortive proteasome activities. The third was cytoplasmic foci, which appeared regardless of abortive proteasome activities. Interestingly, ubiquitin, but not SUMO-2/3, might play a role in assembling cytoplasmic OP-Puropositive foci, and promiscuous SUMO-2/3 with ubiquitin might contribute to OP-Puro assembly at PML-NBs, but not OP-Puro accumulation at the nucleolus. These findings indicate for the first time the previously unappreciated nuclear trafficking of immature polypeptides and also support the role of PML-NBs and the SUMO/ubiquitin pathway in nuclear protein homeostasis. Given that the nucleus is more prone to protein aggregation than the cytoplasm and that misfolded cytoplasmic proteins seem to be actively imported into the nucleus [18,19], our findings support the idea of the intra-nuclear sequestration of aberrant proteins to provide protection against their cytotoxicity [20e22]. Conflict of interest The authors declare no conflicts of interest. Acknowledgments 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). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.03.155.
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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] S.R. Starck, H.M. Green, J. Alberola-Ila, R.W. Roberts, A general approach to detect protein expression in vivo using fluorescent puromycin conjugates, Chem. Biol. 11 (2004) 999e1008. [5] 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. [6] 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. [7] 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. [8] A. David, J.W. Yewdell, Applying the ribopuromycylation method to detect nuclear translation, Methods Mol. Biol. 1228 (2015) 133e142. [9] R. Bernardi, P.P. Pandolfi, Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies, Nat. Rev. Mol. Cell Biol. 8 (2007) 1006e1016. [10] C. Boutell, R.D. Everett, Regulation of alphaherpesvirus infections by the ICP0 family of proteins, J. Gen. Virol. 94 (2013) 465e481. , PML nuclear bodies: regulation, [11] U. Sahin, V. Lallemand-Breitenbach, H. de The function and therapeutic perspectives, J. Pathol. 234 (2014) 289e291. [12] 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 [13] U. Sahin, H. de The and oxidative stress-sensitive sumoylation, Nucleus 5 (2014) 499e507. [14] 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) 83e88. [15] 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. [16] F. Wang, L.A. Durfee, J.M. Huibregtse, A cotranslational ubiquitination pathway for quality control of misfolded proteins, Mol. Cell 50 (2013) 368e378. [17] F. Mongelard, P. Bouvet, Nucleolin: a multiFACeTed protein, Trends Cell Biol. 17 (2007) 80e86. [18] M.S. Hipp, S.H. Park, F.U. Hartl, Proteostasis impairment in protein-misfolding and -aggregation diseases, Trends Cell Biol. 24 (2014) 506e514. [19] A.C. Woerner, F. Frottin, D. Hornburg, L.R. Feng, F. Meissner, M. Patra, J. Tatzelt, M. Mann, K.F. Winklhofer, F.U. Hartl, M.S. Hipp, Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA, Science 351 (2016) 173e176. [20] F. Saudou, S. Finkbeiner, D. Devys, M.E. Greenberg, Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions, Cell 5 (1998) 55e66. [21] H. Saitoh, J. Hinchey, Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3, J. Biol. Chem. 275 (2000) 6252e6258. [22] L. Guo, B.L. 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.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities Naoki Uozumi a, 1, Hotaru Matsumoto a, 1, 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 23 March 2016 Accepted 30 March 2016 Available online 25 April 2016
The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycinconjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis. © 2016 Elsevier Inc. All rights reserved.
Keywords: Protein homeostasis Puromycin Click chemistry Small ubiquitin-related modifier (SUMO) Ubiquitin Promyelocytic leukemia nuclear bodies
1. Introduction Puromycin, an amino-nucleoside antibiotic produced by Streptomyces alboniger, is a potent protein synthesis inhibitor in both prokaryotic and eukaryotic cells [1]. Puromycin enters the ribosome A site and terminates translation by ribosome-catalyzed covalent incorporation into the nascent polypeptide chain, referred to as ‘puromycylation’ [2e8]. Because this generates prematurely terminated and misfolded polypeptides, puromycin-conjugated (puromycylated) polypeptides are likely to cause cytotoxic effects and thus may contribute to induction of the stress response and growth defects; however, regulation of their trafficking and fate in vital cellular systems remain largely uncharacterized. Promyelocytic leukemia-nuclear bodies (PML-NBs), also known
Abbreviations: OP-Puro, O-propargyl-puromycin; SUMO, small ubiquitin-related modifier; PML-NBs, promyelocytic leukemia-nuclear bodies; DAPI, 40 ,6-diamidino2-phenylindole. * 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). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.03.155 0006-291X/© 2016 Elsevier Inc. All rights reserved.
as nuclear dot 10 (ND10) or PML oncogenic domains (PODs), are nuclear domains characterized by the presence of the tripartite motif family protein PML/TRIM19 [9e13]. PML-NBs are involved in a wide variety of cellular activities, including the stress response, aging and viral infection [9e13]. A wide variety of proteins are assembled into PML-NBs via post-translational modification of PML and/or its interactors mediated by the SUMO-2/3 and ubiquitin conjugation pathways. This may contribute to a protective response involving the intra-nuclear sequestration, storage or degradation of non-functional and aberrant proteins [9e13]. Here we use a puromycin derivative, O-propargyl-puromycin (OP-Puro) to monitor puromycylated polypeptides in cultured HeLa cells. OP-Puro is incorporated into elongating polypeptides on ribosomes in a manner similar to puromycin and can be fluorescently labeled by an azide-alkyne reaction, called ‘click chemistry’ [4e8]. This enables the subcellular distribution of OP-puromycylated polypeptides to be visualized in cells. Notably, PO-Puro signals are detected in the cytoplasm and nucleus with substantial concentration at the nucleolus under standard culture conditions. Intriguingly, OP-Puro signals are detected at multiple small nuclear foci when the proteasome inhibitor MG132 is co-administrated. These small nuclear foci may represent PML-NBs, because PML,
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SUMO-2/3 and ubiquitin co-accumulate at the foci. Our findings suggest a role for the nucleus together with the SUMO and ubiquitin pathways in protein homeostasis and the intracellular sequestration of immature polypeptides.
2. Materials and methods 2.1. Cell culture and drug treatments HeLa cells were cultured 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. Protein synthesis inhibitors, puromycin, cycloheximide, emetin and anisomycin, were obtained from Wako Pure Chemical Industries. O-propargyl-puromycin (OP-Puro) was obtained from Jena Bioscience. Benzyloxycarbonyl-L-leucyl-Lleucyl-L-leucinal (MG132) was purchased from Peptide Institute Inc. Each drug was dissolved in dimethyl sulfoxide (DMSO) and added to the culture medium as indicated in the text.
2.2. Antibodies Anti-SUMO-2/3 and -SUMO-1 rabbit monoclonal antibodies were obtained from Cell Signaling Technologies. Anti-polyubiquitin mouse monoclonal (FK2), anti-PML and anti-nucleolin antibodies were obtained from Medical and Biological Laboratories (MBL), Santa Cruz Biotechnology and MerckMillipore, respectively. Secondary antibodies used were purchased from Jackson ImmunoResearch, Life technologies and MBL.
2.3. OP-Puro labeling of cultured HeLa cells and detection by fluorescence microscopy OP-Puro labeling was carried out as previously described [5]. In brief, HeLa cells were grown on glass coverslips in culture medium. To visualize OP-Puro incorporation, we typically exposed cells to culture medium containing 10 mM OP-Puro and 10 mM MG132 for 4 h at 37 C. The cells were washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were washed with PBS, followed by permeabilization with PBS containing 0.1% Triton X-100, and then washed with PBS. OP-Puro was detected by performing an azidealkyne cycloaddition for 30 min at room temperature using the Click-iT Cell Reaction Buffer Kit containing 15 mM Alexa 488/555-N3 azide (Life Technologies). After staining, the coverslips were washed several times with PBS and counterstained with 40 ,6diamidino-2-phenylindole (DAPI). If indirect-immunofluorescence was performed using specific antibodies to co-localize OP-Puro signals, the cells were incubated with the appropriate antibody and the indirect-immunofluorescence reaction continued as described below. Fluorescence images were obtained using a DP72 microscope (Olympus).
2.4. Indirect-immunofluorescence analysis Indirect-immunofluorescence was performed as described previously [14,15]. Briefly, HeLa cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were blocked in PBS containing 0.2% bovine
Fig. 1. OP-Puro signals accumulated in the nucleus under standard conditions, and were enhanced during abortive proteasome activities. (A) Schematic representation of the effects of OP-Puro/puromycin. OP-Puro/puromycin, leads to premature release of nascent polypeptides by puromycylation and thus inhibiting polypeptide chain elongation on the ribosome and releasing puromycylated immature polypeptides. In the case of OP-puromycylated polypeptides, their subcellular localization can be visualized by click reaction with fluorescence-conjugated N3. B) Exponentially growing HeLa cells under standard culture conditions were exposed to 10 mM Puro in the absence (upper panels) or presence (bottom panels) of 10 mM MG132 for the indicated periods at 37 C, and were then subjected to the click reaction using Alexa 488-N3 to visualize OP-Puro localization. Bar indicates 20 mm.
N. Uozumi et al. / Biochemical and Biophysical Research Communications 474 (2016) 247e251
serum albumin, 0.1% Triton X-100 for 20 min and incubated with primary antibody for 1 h, followed by an appropriate secondary antibody. The coverslips were analyzed with microscope. DNA was visualized with DAPI.
3. Results and discussion 3.1. Detection of OP-Puro-positive nuclear foci in HeLa cells To elucidate OP-Puro localization in cultured HeLa cells, we exposed cells to OP-Puro for 0, 3, 10, 30, 90 and 240 min, followed by fixation and ‘click chemistry’ (Fig. 1A and B). OP-Puro staining under our experimental conditions for short incubation periods (3e10 min) produced very similar staining patterns to those described previously by several groups [4e8]; signals appeared after incubation for as little as 3 min and the signals were distributed diffusely throughout the cell. Intriguingly, after incubation for 10 min, OP-Puro stained large foci (diameter >1 mm) in the nucleus. In many cells, approximately two to four large OP-Puro-stained foci were detected in nuclei (Fig. 1A, B and Supplementary Fig. 1). We also observed multiple OP-Puro-positive foci in the cytoplasm when cells were exposed to OP-Puro for longer periods (90e240 min; Fig. 1A, B and Supplementary Fig. 1). It should be noted that neither nuclear nor cytoplasmic OP-Puro-positive staining were visible when ribosome-binding translation inhibitors, such as cycloheximide, emetine or anisomycin, were co-
249
administrated with OP-Puro (Supplementary Fig. 2), indicating that OP-Puro signals were not due to non-specific products of click chemistry and that the signals represented products associated with ribosome-mediated translation activity. Puromycylated polypeptides are subject to rapid degradation via the ubiquitin-proteasome system (UPS) [16]; therefore, we predicted that OP-Puro signals would be enhanced if proteasome activities were inhibited. As expected, OP-Puro staining was enhanced in cells during abortive proteasome activities. Additionally, we found that multiple small OP-Puro-positive foci (diameter < 1 mm) became visible in the nucleus (Fig. 1A, B and Supplementary Fig. 1), suggesting the existence of nuclear spots where degradation-defective, puromycylated immature polypeptides were accumulated. Taken together, our results indicated that, during longer exposure to OP-Puro and abortive proteasome activities, OP-Puro signals were not randomly distributed throughout the cell; indeed there were multiple regions which facilitated OP-Puro accumulation in the nucleus.
3.2. OP-Puro-positive large and small nuclear foci represented the nucleolus and PML-nuclear bodies, respectively To elucidate the functions of OP-Puro-positive foci in the nucleus, we aimed to identify the components associated with these foci using specific antibodies. To this end, indirectimmunofluorescence using antibodies against nucleolin, a well-
Fig. 2. Nuclear OP-Puro preferentially accumulated in the nucleolus and PML-NBs. (A) Hela cells were exposed to 10 mM Puro in the absence (left column) or presence (right column) of 10 mM MG132 for 240 min at 37 C, and were then subjected to the click reaction using Alexa 488-N3 (upper panels) and immunostaining using an anti-nucleolin antibody (upper-middle panels). Merged images of click reaction and nucleolin-staining are shown in the lower-middle panel. DNA was visualized with DAPI staining (bottom panels). Bar indicates 20 mm. (B) Hela cells were exposed to 10 mM Puro in the absence (left column) or presence (right column) of 10 mM MG132 for 240 min at 37 C, and were then subjected to the click reaction using Alexa 488-N3 (upper panels) and immunostaining using an anti-PML antibody (upper-middle panels). Merged images of click reaction and nucleolin-staining are shown in the lower-middle panel. DNA was visualized with DAPI staining (bottom panels). Bar indicates 20 mm.
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Fig. 3. SUMO-2/3 and ubiquitin were merged with OP-Puro staining at small nuclear foci. (A) Hela cells were exposed to no drug (upper), 10 mM OP-Puro (upper-middle), 10 mM MG132 (lower-middle) or 10 mM OP-Puro plus 10 mM MG132 (lower) for 240 min at 37 C, and were then subjected to the click reaction using Alexa 488-N3 (left column) and immunostaining using an anti-SUMO-2/3 antibody (center column). Merged images are shown at the right. (B) Hela cells were exposed to no drug (upper), 10 mM OP-Puro (uppermiddle), 10 mM MG132 (lower-middle) or 10 mM OP-Puro plus 10 mM MG132 (lower) for 240 min at 37 C, and were then subjected to the click reaction using Alexa 555-N3 (left column) and immunostaining using an anti-FK2 ubiquitin antibody (center column). Merged images are shown at the right. (C) Subcellular OP-Puro distribution detected by click chemistry which indicates a putative trafficking pathway of puromycylated immature polypeptides. OP-Puro signals are distributed in the cytoplasm and nucleoplasm. After longer incubation with OP-Puro, the signals tend to form foci in the cytoplasm and to accumulate at the nucleolus in the nucleoplasm. When proteasome activities are aborted by MG132, OP-Puro signals accumulate at PML-NBs together with SUMO and ubiquitin.
known nucleolus-marker protein [17], revealed co-staining with OP-Puro signals at large nuclear foci (Fig. 2A). Additionally, indirectimmunofluorescence using antibodies against puromyelocytic leukemia (PML), a PML-nuclear body (PML-NB) component [9e11], indicated co-localization of PML with small OP-Puro-positive nuclear foci (Fig. 2B). These results suggested that there were two different-types of nuclear compartment where OP-Puro signals accumulated; the large OP-Puro-positive nuclear foci, which were detected both in the absence and presence of MG132, represented the nucleolus, and the small OP-Puro-positive nuclear foci, which appeared only in the presence of MG132, represented PML-NBs. It should be noted that many, but not all, small OP-Puro-positive
nuclear foci merged with PML-staining (Fig. 2B), indicating the existence of unidentified nuclear bodies that contained puromycylated immature polypeptides. 3.3. OP-Puro, SUMO and ubiquitin were promiscuously accumulated at PML-NBs during abortive proteasome activities The SUMO-2/3 and ubiquitin pathways participate in the regulation of PML function and PML-NB formation; therefore, we suspected involvement of these modification pathways for OP-Puro assembly at PML-NBs during abortive proteasome activities. As shown in Fig. 3, anti-SUMO-2/3 antibody and-ubiquitin (FK2)
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antibodies, clearly stained small OP-Puro-positive nuclear foci only in cells exposed to OP-Puro and MG132, whereas neither SUMO-2/3 nor ubiquitin staining was merged with OP-Puro staining in the absence of MG132. These results suggested that promiscuous SUMO and ubiquitin assemblies were induced together with the accumulation of OP-Puro signals at PML-NBs, but not at the nucleolus, during abortive proteasome activities. Of note, we found that the ubiquitin signals merged with cytoplasmic foci, indicating involvement of the ubiquitin pathway in the formation of OP-Puropositive cytoplasmic foci. Taken together, we revealed that there were at least three cellular compartments where OP-Puro tended to accumulate (Fig. 3C); one was larger nuclear domains, representing the nucleolus, which were observed both in the absence and presence of MG132. The second was multiple small nuclear foci, representing PML-NBs, which were detectable during abortive proteasome activities. The third was cytoplasmic foci, which appeared regardless of abortive proteasome activities. Interestingly, ubiquitin, but not SUMO-2/3, might play a role in assembling cytoplasmic OP-Puropositive foci, and promiscuous SUMO-2/3 with ubiquitin might contribute to OP-Puro assembly at PML-NBs, but not OP-Puro accumulation at the nucleolus. These findings indicate for the first time the previously unappreciated nuclear trafficking of immature polypeptides and also support the role of PML-NBs and the SUMO/ubiquitin pathway in nuclear protein homeostasis. Given that the nucleus is more prone to protein aggregation than the cytoplasm and that misfolded cytoplasmic proteins seem to be actively imported into the nucleus [18,19], our findings support the idea of the intra-nuclear sequestration of aberrant proteins to provide protection against their cytotoxicity [20e22]. Conflict of interest The authors declare no conflicts of interest. Acknowledgments 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). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.03.155.
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