Letter pubs.acs.org/synthbio
Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX
Huntingtin Polyglutamine-Dependent Protein Aggregation in Reconstituted Cells Kodai Machida,*,†,‡ Kuru Kanzawa,† Tomoaki Shigeta,† Yuki Yamamoto,† Kanta Tsumoto,§ and Hiroaki Imataka*,†,‡ †
Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan § Division of Chemistry for Materials, Graduate School of Engineering, Mie University, Tsu 514-8507, Japan ‡
S Supporting Information *
ABSTRACT: One of the aims of synthetic biology is bottom-up construction of reconstituted human cells for medical uses. To that end, we generated giant unilamellar vesicles (GUVs) that contained a HeLa cell extract, which comprises a cell-free protein synthesis (CFPS) system. Then we expressed Huntingtin protein fragments that contained polyglutamine (polyQ) sequences (Htt-polyQ), a hallmark of Huntington’s disease. That system produced polyQ-dependent protein aggregates, as previously demonstrated in living cells. We next simplified the system by generating GUVs that contained purified human factors, which reconstituted a CFPS system. Htt-polyQ fragments expressed in these GUVs also formed protein aggregates. Moreover, an N-terminal deletion mutant, which had failed to form protein aggregates in living cells, also failed to form protein aggregates in the reconstituted GUVs. Thus, the GUV systems that encapsulated a human CFPS system could serve as reconstituted cells for studying neurological diseases. KEYWORDS: cell-free protein synthesis, GUV, Huntingtin, polyglutamine, protein aggregation, reconstitution
C
One of the hallmarks of neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease is the accumulation of protein aggregates in neuronal cells. Huntington’s disease is caused by a mutant Huntingtin (Htt) protein, but the normal function of this protein remains obscure. Patients with Huntington’s disease harbor Htt proteins with multiple, sequential glutamine residues at the N-terminus, and the length of this polyglutamime (polyQ) sequence is correlated with the strength of its proclivity for aggregation.14,15 We previously established a cell-free assay system for studying polyQ-dependent protein aggregation with a HeLa cell extract-derived CFPS system.16 In that system, we synthesized polyQ sequences fused to enhanced green fluorescent protein (polyQ-EGFP), and when these polyQ-EGFP products were passed through a filter membrane, polyQ-dependent aggregates could be trapped on the membrane.16 In this study, we generated GUVs that encapsulated a CFPS system, which consisted of either a HeLa cell extract or purified human factors, and synthesized polyQ Htt fragments. These systems faithfully recapitulated protein aggregation observed in living cells. Therefore, these systems represented
ell-free protein synthesis (CFPS) systems are important tools for producing recombinant proteins, and also, for mechanistic analyses of translation and post-translational events. CFPS systems are divided into two types: cell extract-based systems1−4 and reconstituted systems.5,6 Cell extract-derived CFPS systems consist of the cytoplasm from disrupted cells; therefore, they contain most cellular constituents, except the nucleus (or DNA) and the plasma membrane (and/or cell wall). In contrast, reconstituted systems comprise limited components that are required for translation and/or post-translation. In both systems, an RNA polymerase can transcribe mRNAs from a DNA template placed in the same test tube, which thereby recapitulates the central dogma of biology. Reconstituted cells or cell-like containers have been devised by encapsulating a CFPS system in liposome or giant unilamella vesicles (GUVs). For example, S30, a bacterial extract-based CFPS, was encapsulated in liposomes or GUVs, and various proteins were expressed therein.7,8 The GUV encapsulation of the PURE system, a CFPS system reconstituted from bacterial components6 has led to studies on the integration of membrane proteins9,10 and to novel applications, such as the in vitro evolution of aminoacyl-tRNA synthetase,11 membrane proteins12 and alpha-hemolysin.13 © XXXX American Chemical Society
Received: October 16, 2017 Published: December 12, 2017 A
DOI: 10.1021/acssynbio.7b00372 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology
Figure 1. Schematic illustration of the expression vectors and the experimental procedure.
useful in vitro assays for analyzing human neurological diseases.
■
RESULTS AND DISCUSSION As a first step in establishing an in vitro system for studying neurodegenerative diseases, we previously devised a cell-free system with a HeLa cell extract-derived CFPS for analyzing polyQ-dependent protein aggregation.16 When a polyQ-EGFP protein, N17-polyQ-PRD-EGFP-HA, which contained the Htt N-terminal 17 amino acids (N17),17 followed by the polyQ domain, the Htt proline rich domain (PRD),17 EGFP, and a hemagglutinin (HA) tag (Figure 1) was produced in the HeLa cell extract-derived CFPS system, protein aggregates formed that could not permeate a filter membrane.16 In living cells, the same protein was visualized as fluorescent dots16 that represented protein aggregates (see Figures 5 and 6 of the present paper). Thus, the filter-trap assay appeared to reproduce protein aggregation in cells. However, it remained to be rigorously explored whether the aggregates formed in vitro would correspond to the protein aggregates in living cells, because, in principle, the assay systems are different. Therefore, we reasoned that performing polyQ-dependent aggregation analyses in reconstituted cells that contained a human CFPS system might provide data that better reflected in vivo phenomena. Here, we encapsulated the HeLa cell extract-derived CFPS in GUVs and expressed one of three different constructs therein: N17-96Q-PRD-EGFP-HA, N17-25Q-PRD-EGFP-HA, or EGFP-HA, which served as a control (Figure 1). When EGFP-HA and N17-25Q-PRD-EGFP-HA were expressed in GUVs, EGFP fluorescence was uniformly distributed in GUVs (Figure 2), as shown previously in HeLa cells that expressed these proteins.16 When the 96Q-containing EGFP was expressed, dot-like fluorescence was observed in GUVs (Figure 2), similar to observations in HeLa cells. 16 Preinclusion of chaperonin CCT or the Hsc70 chaperone system (Hsc70, Hsp40 and Hsp110)16 impeded the formation of aggregate dots (Figure 3), consistent with the previous findings indicating that expressing these chaperone proteins reduced polyQ-dependent protein aggregation in cultured cells or animals.17−20 Thus, the GUV system that encapsu-
Figure 2. Confocal microscopy images show distributions of proteins expressed in liposomes (GUVs) with the HeLa extract-derived CFPS system. Distributions of EGFP-HA (control) and Htt polyQmodified proteins, N17-25Q-PRD-EGFP-HA and N17-96Q-PRDEGFP-HA, are shown in bright-field (BF) and fluorescence (GFP) images, captured after 20-h incubations at 32 °C.
lated HeLa cell extracts faithfully reproduced the polyQdependent protein aggregation observed in living cells. Cell extracts contain numerous proteins and other cellular constituents, which makes it difficult to examine the molecular mechanism of the polyQ-dependent protein aggregation. Thus, we decided to create GUVs that encapsulated a translation system reconstituted with human components. Originally, we reconstituted a CFPS system with ribosomes, tRNAs, elongation factors, termination factors and aminoacyl tRNA synthetases. When an HCV IRES-dependent expression vector was used as a template, the ribosome bound directly to the HCV IRES, and hence the system did not require eukaryotic translation initiation factors (eIFs).5 However, this system showed relatively low productivity; when this reconstituted system was encapsulated in GUVs and B
DOI: 10.1021/acssynbio.7b00372 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology
Figure 3. Chaperone proteins inhibited polyQ-dependent aggregation in GUVs. (a) Confocal fluorescence (GFP) images of GUVs, which contained the HeLa extract-derived CFPS system and expressed N17-96Q-PRD-EGFP-HA. The system was presupplemented with CCT (5 μM) or Hsc70s (5 μM)16 before incubation. Images were acquired at 20 h (top row) and 44 h (bottom row) of incubation. (b) Quantitative analysis of the percentage of GUVs that harbored aggregation dots among the total GUVs observed in each image. Calculations were performed after 20 h (Black columns), 30 h (gray columns), and 44 h (white columns) of incubation. Each column and bar represents the average and standard deviation of three images, respectively.
finding suggests that the Htt protein tends to aggregate intrinsically in cells, without the assistance of other cellular proteins, except translation-related factors. Then we examined whether CCT or Hsp70s would prevent the Htt protein aggregation in this system. When N17-96Q-PRD-EGFP-HA was expressed in the reconstituted GUVs with or without CCT or Hsc70s, no noticeable effects of these chaperones were observed (Supplementary Figure S1), suggesting that CCT or Hsc70s could impede the Htt protein aggregation in cooperation with other factors present in the cytoplasm. We further demonstrated the validity of this system by examining the role of the N-terminal sequence in the Htt fragment. The N-terminal 17-amino acid sequence in front of the polyQ stretch in the Htt fragment was previously shown to be essential for aggregation.17,22−25 As expected, when the Htt fragment lacking the N-terminal sequence (96Q-PRDEGFP-HA) (Figure 1) was expressed in HeLa cells, no fluorescent dots were formed (Figure 6). Likewise, when the same protein was expressed in the reconstituted GUVs, we detected uniform fluorescence in GUVs, without the fluorescent dots observed when the complete Htt fragment (N17-96Q-PRD-EGFP-HA) was expressed (Figure 6). In conclusion, our results showed that a system of reconstituted cells (GUVs) could reproduce the Htt-polyQ aggregation observed in living cells; therefore, this system might be useful for analyzing protein aggregation observed in neurological diseases. A future project could use this system to explore the mechanism of repeat-associated non-ATG (RAN) translation.26 Briefly, in addition to polyQ, both polyserine and polyalanine aggregates were detected in autopsy tissues from patients with Huntington disease. The latter two aggregates resulted from the translation of repeated AGC and GCA sequences, which encoded polyserines and polyalanines, respectively. Interestingly, both these open reading frames started with non-AUG codons for translation initiation.26 Our reconstituted GUV system of human translation factors may be an effective tool for studying RAN translation, which provokes potentially harmful protein aggregation.
programmed with HCV EGFP-HA, very faint fluorescence signals were produced (Figure 4b). Therefore, we examined whether adding a translation factor might increase the productivity of this CFPS system. We found that adding eIF2 and ABCE1 enhanced translation (Figure 4a). This enhancement was probably achieved by the action of eIF2, which efficiently accommodated the methionyl initiator tRNA to the ribosome, and thus, promoted translation initiation; in addition, ABCE1 contributed by promoting ribosome recycling.21 The addition of other translation factors had no effect. Note that ABCE1 also enhances translation in a reconstituted 5′-cap-dependent system (H. Imataka, unpublished data), suggesting that this recycling factor is required for efficient translation in general. When EGFP-HA was expressed in GUVs that encapsulated the reconstituted system and included both eIF2 and ABCE1, notable EGFP fluorescence was observed (Figure 4b). Next, we encapsulated the improved reconstituted CFPS system in GUVs, and expressed the polyQ-EGFP protein therein. N17-96Q-PRD-EGFP-HA formed aggregates (detected as dots) in GUVs, similar to the observation in transfected HeLa cells (Figure 5). In contrast, the EGFP protein (control) was uniformly distributed in GUVs. This
METHODS Plasmids. DNA sequences complementary to the human ABCE1 (acc. No. NM_002940) and NFS1 (NM_001198989) genes were amplified from human placenta RNA (Clontech) with reverse transcription (RT)-PCR. ABCE1 cDNA was cloned into the pCIneo vector (Promega) with triple FLAG sequences attached to the C-terminal sequence to generate the pCIneo ABCE1−3xFLAG construct. NFS1 cDNA was cloned into pCIneo to generate the pCIneo NFS1 construct. DNA sequences complementary to the human eIF2α (NM_004094), eIF2β (NM_003908), eIF2γ (NM_001415), and cdc123 (NM_006023) genes were amplified from HeLacell poly(A) RNA with RT-PCR. These four cDNAs were cloned into pCIneo vectors. One FLAG sequence was attached to the N-terminal sequence of eIF2γ to generate the pCIneo FLAG eIF2γ construct. Recombinant Proteins. ABCE1: the pCIneo ABCE1− 3xFLAG plasmid was cotransfected into HEK293T cells with the pCIneo NFS1 plasmid to facilitate [Fe−S] cluster formation in the ABCE1 protein.27 Recombinant ABCE1− 3xFLAG protein was purified from cytoplasmic extracts of transfected cells with anti-FLAG M2 agarose chromatography (SIGMA).
■
C
DOI: 10.1021/acssynbio.7b00372 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology
Figure 4. ABCE1 and eIF2 improved performance of the reconstituted CFPS system. (a, left panel) Luciferase activity (arbitrary units) shows translation levels achieved after the reconstituted CFPS system was incubated with the pUC HCV HA-Renilla luciferase,5 in the presence or absence of ABCE1 and/or eIF2 (0.5 μM each) for 1.5 to 24 h. (a, right panel) ABCE1 (1.0 μg) and eIF2 (1.5 μg) stained with Coomassie Brilliant Blue. (b, left panel) Confocal microscopy images (BF: bright-field; GFP: fluorescence) of GUVs that contained the reconstituted CFPS system and expressed EGFP-HA. Images show expression in the presence and absence of ABCE1 + eIF2 after 20-h incubations. (b, right panel) Western blot probed with the anti-HA antibody shows the EGFP-HA protein levels expressed in GUVs with and without added enhancement factors. Each protein band of EGFP-HA was quantified using ImageJ 1.48v software (https://imagej.nih.gov/ij/), and the relative values were reported below each lane.
mL, Avanti: 850457C) and cholesterol (10 mg/mL, Nacalai: 08722−81), both dissolved in chloroform (Wako: 038− 02606), were mixed at a 1:1 (v/v) ratio in a 1.7-ml test tube (BMBio: BM4017). The POPC-cholesterol mixture was then mixed with liquid paraffin (Wako: 128−04375) at a 1:10 (v/ v) ratio in the test tube. The POPC-cholesterol-paraffin mixture was incubated for 30 min at 80 °C to vaporize the chloroform; then, the mixture was cooled on ice until use. The “inner solution” (10 μL) comprised the CFPS reaction mixture and 200 mM sucrose. This solution was added to the POPC-cholesterol-paraffin mixture (50 μL) and mixed by tapping the bottom of the test tube to generate the water-inoil emulsion. The emulsion (60 μL) was layered onto the outer buffer (60 μL), which comprised the CFPS reaction buffer, containing NTPs, 20 amino acids, and 200 mM glucose. The test tube was incubated for 10 min on ice, then centrifuged at 10 000g for 30 min at 4 °C. The liposome suspension was harvested from the bottom of the test tube. The liposome suspension was incubated for 20−48 h at 32 °C for protein synthesis. Analysis of Aggregates in GUVs. Bright-field (BF) and fluorescence (GFP) images of liposomes were viewed with a confocal laser scanning microscope (Carl Zeiss: LSM510). The observation chamber consisted of a silicone rubber sheet (Kenis: 5HL3−604, 0.1- mm thick) with a hole (6 mm diameter), placed on a glass microscope slide (MATSUNAMI:
eIF2: the eIF2 expression plasmids (pCIneo eIF2α, pCIneo eIF2β, and pCIneo FLAG eIF2γ) were cotransfected into HEK293T cells with the pCIneo cdc123 plasmid to facilitate formation of the eIF2 complex.28 Recombinant eIF2 was purified from cytoplasmic extracts of transfected cells with anti-FLAG M2 agarose chromatography, followed by QSepharose chromatography (GE Healthcare). CFPS Systems. The HeLa cell extract-derived CFPS system was prepared as described previously.3 The CFPS system reconstituted with human factors5 was modified as follows. The system was supplemented with ABCE1 and eIF2 recombinant proteins, and the final concentration of glycerol in the reaction mixture was set to less than 1.5%. This modified translation system contained: 1 mg/mL nativetRNAs, 50 μM recombinant eEF1 complex (eEF1A, eEF1Bα, and eEF1Bγ), 0.5 μM 40S ribosome, 0.5 μM 60S ribosome, 1 μM native eEF2, 0.5 μM recombinant eRF1/3 mixture, 100 μM of a 20-amino acid mixture (Promega), 150 ng/μL of a recombinant aminoacyl tRNA synthetase mixture, 0.01 mg/ mL recombinant T7 RNA polymerase, 12.5 ng/μL plasmid DNA, 0.1 μM recombinant PPA1, 0.5 μM recombinant ABCE1, and 0.5 μM recombinant eIF2, in a total volume of 10 μL. Formation of GUVs. Liposomes (GUVs) were produced with the water-in-oil emulsion transfer method previously described,12 with some modifications. Briefly, POPC (10 mg/ D
DOI: 10.1021/acssynbio.7b00372 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology
Figure 5. Reconstituted GUVs reproduced polyQ-dependent protein aggregation. (Upper panels) Confocal bright field (BF) and fluorescence (GFP) images of GUVs that contained the improved reconstituted CFPS system and expressed EGFP-HA (control) or N17-96Q-PRD-EGFPHA. Three different microscopic fields are shown for each protein. Observations were performed after 20-h incubations. (Lower panels) The same proteins were expressed for 12 h in HeLa cells.
Figure 6. Importance of the N-terminal sequence of the Htt fragment. (Upper panels) Confocal bright field (BF) and fluorescence (GFP) images of GUVs that contained the improved reconstituted CFPS system and expressed 96Q-PRD-EGFP-HA or N17-96Q-PRD-EGFP-HA. Two different microscopic fields are shown for each protein. Observations were performed after 20 h of incubation. (Lower panels) The same proteins were expressed for 12 h in HeLa cells.
S111). The sample (3 μL) was applied to the hole and covered with a glass microscope coverslip (MATSUNAMI: 24 mm × 24 mm, thickness NO. 1, 0.13−0.17 mm). The
observations were performed at room temperature, and the pinhole size was fixed at 5 μm. EGFP was excited with an argon laser at 488 nm, and the fluorescence images were E
DOI: 10.1021/acssynbio.7b00372 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology acquired through a LP505 emission filter. The images were analyzed with ImageJ-1.48v software. Cell Culture and Transfection. HeLa cells were cultured to 50% confluence in DMEM supplemented with 10% fetal calf serum, 1% L-alanyl-L-glutamine solution, and 1% penicillin-streptomycin in 6-well plates. The cells in each well were transfected with 2 μg of plasmid (pCI-neo-EGFPHA, pCI-neo-N17-25Q-PRD-EGFP-HA, pCI-neo-N17-96QPRD-EGFP-HA, or pCI-neo-96Q-PRD-EGFP-HA)16 and Lipofectamine 2000 (6 μL) in OPTI-MEM solution (600 μL). Transfected cells were incubated for 3 h in a CO2-gassed incubator; then 2 mL of DMEM, supplemented with 10% FCS, was added, and the cells were incubated for another 12 h. Transfected cells were observed with a CKX41 inverted microscope (Olympus), as described previously.16 Luciferase Activity. The enzyme activity of renilla luciferase, synthesized in the reconstituted CFPS system, was measured with a Dual-Luciferase Reporter (DLR) Assay System (Promega: E1910). The signal was evaluated with a Lumat LB9507 luminometer (EG&G Berthold).
■
proteins 2A and 2B occurs in the elongation phase of translation without eukaryotic release factors. J. Biol. Chem. 289, 31960−31971. (6) Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. (2001) Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751− 755. (7) Noireaux, V., and Libchaber, A. (2004) A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. U. S. A. 101, 17669−17674. (8) Nomura, S. M., Tsumoto, K., Hamada, T., Akiyoshi, K., Nakatani, Y., and Yoshikawa, K. (2003) Gene expression within cellsized lipid vesicles. ChemBioChem 4, 1172−1175. (9) Kuruma, Y., Stano, P., Ueda, T., and Luisi, P. L. (2009) A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. Biochim. Biophys. Acta, Biomembr. 1788, 567−574. (10) Soga, H., Fujii, S., Yomo, T., Kato, Y., Watanabe, H., and Matsuura, T. (2014) In vitro membrane protein synthesis inside cellsized vesicles reveals the dependence of membrane protein integration on vesicle volume. ACS Synth. Biol. 3, 372−379. (11) Uyeda, A., Watanabe, T., Kato, Y., Watanabe, H., Yomo, T., Hohsaka, T., and Matsuura, T. (2015) Liposome-Based in Vitro Evolution of Aminoacyl-tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation. ChemBioChem 16, 1797−1802. (12) Fujii, S., Matsuura, T., Sunami, T., Nishikawa, T., Kazuta, Y., and Yomo, T. (2014) Liposome display for in vitro selection and evolution of membrane proteins. Nat. Protoc. 9, 1578−1591. (13) Fujii, S., Matsuura, T., Sunami, T., Kazuta, Y., and Yomo, T. (2013) In vitro evolution of alpha-hemolysin using a liposome display. Proc. Natl. Acad. Sci. U. S. A. 110, 16796−16801. (14) Penney, J. B., Jr., Vonsattel, J. P., MacDonald, M. E., Gusella, J. F., and Myers, R. H. (1997) CAG repeat number governs the development rate of pathology in Huntington’s disease. Ann. Neurol. 41, 689−692. (15) Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G. P., Davies, S. W., Lehrach, H., and Wanker, E. E. (1997) Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90, 549−558. (16) Machida, K., Shigeta, T., Kobayashi, A., Masumoto, A., Hidaka, Y., and Imataka, H. (2016) Cell-free analysis of polyQ-dependent protein aggregation and its inhibition by chaperone proteins. J. Biotechnol. 239, 1−8. (17) Tam, S., Geller, R., Spiess, C., and Frydman, J. (2006) The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat. Cell Biol. 8, 1155−1162. (18) Kitamura, A., Kubota, H., Pack, C. G., Matsumoto, G., Hirayama, S., Takahashi, Y., Kimura, H., Kinjo, M., Morimoto, R. I., and Nagata, K. (2006) Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state. Nat. Cell Biol. 8, 1163− 1170. (19) Muchowski, P. J., Schaffar, G., Sittler, A., Wanker, E. E., HayerHartl, M. K., and Hartl, F. U. (2000) Hsp70 and hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils. Proc. Natl. Acad. Sci. U. S. A. 97, 7841−7846. (20) Satyal, S. H., Schmidt, E., Kitagawa, K., Sondheimer, N., Lindquist, S., Kramer, J. M., and Morimoto, R. I. (2000) Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 97, 5750−5755. (21) Pisarev, A. V., Skabkin, M. A., Pisareva, V. P., Skabkina, O. V., Rakotondrafara, A. M., Hentze, M. W., Hellen, C. U., and Pestova, T. V. (2010) The role of ABCE1 in eukaryotic posttermination ribosomal recycling. Mol. Cell 37, 196−210. (22) Caron, N. S., Desmond, C. R., Xia, J., and Truant, R. (2013) Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin. Proc. Natl. Acad. Sci. U. S. A. 110, 14610−14615. (23) Crick, S. L., Ruff, K. M., Garai, K., Frieden, C., and Pappu, R. V. (2013) Unmasking the roles of N- and C-terminal flanking
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00372. Figure S1 (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Hiroaki Imataka: 0000-0002-3731-0294 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Drs. Yoshihisa and Yokota for the use of the confocal fluorescence microscope. This work was supported by JSPS KAKENHI Grants (JP26830141 and 17H04995) awarded to K.M and by JSPS KAKENHI Grants (JP15H04324 and JP20112006) awarded to H.I.
■
REFERENCES
(1) Jewett, M. C., and Swartz, J. R. (2004) Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol. Bioeng. 86, 19−26. (2) Kigawa, T., Yabuki, T., Matsuda, N., Matsuda, T., Nakajima, R., Tanaka, A., and Yokoyama, S. (2004) Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 63−68. (3) Mikami, S., Kobayashi, T., Masutani, M., Yokoyama, S., and Imataka, H. (2008) A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expression Purif. 62, 190−198. (4) Sawasaki, T., Hasegawa, Y., Morishita, R., Seki, M., Shinozaki, K., and Endo, Y. (2004) Genome-scale, biochemical annotation method based on the wheat germ cell-free protein synthesis system. Phytochemistry 65, 1549−1555. (5) Machida, K., Mikami, S., Masutani, M., Mishima, K., Kobayashi, T., and Imataka, H. (2014) A translation system reconstituted with human factors proves that processing of encephalomyocarditis virus F
DOI: 10.1021/acssynbio.7b00372 ACS Synth. Biol. XXXX, XXX, XXX−XXX
Letter
ACS Synthetic Biology sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U. S. A. 110, 20075−20080. (24) Duennwald, M. L., Jagadish, S., Muchowski, P. J., and Lindquist, S. (2006) Flanking sequences profoundly alter polyglutamine toxicity in yeast. Proc. Natl. Acad. Sci. U. S. A. 103, 11045− 11050. (25) Shen, K., Calamini, B., Fauerbach, J. A., Ma, B., Shahmoradian, S. H., Serrano Lachapel, I. L., Chiu, W., Lo, D. C., and Frydman, J. (2016) Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract. eLife, DOI: 10.7554/eLife.18065. (26) Banez-Coronel, M., Ayhan, F., Tarabochia, A. D., Zu, T., Perez, B. A., Tusi, S. K., Pletnikova, O., Borchelt, D. R., Ross, C. A., Margolis, R. L., Yachnis, A. T., Troncoso, J. C., and Ranum, L. P. (2015) RAN Translation in Huntington Disease. Neuron 88, 667− 677. (27) Karcher, A., Schele, A., and Hopfner, K. P. (2008) X-ray structure of the complete ABC enzyme ABCE1 from Pyrococcus abyssi. J. Biol. Chem. 283, 7962−7971. (28) Perzlmaier, A. F., Richter, F., and Seufert, W. (2013) Translation initiation requires cell division cycle 123 (Cdc123) to facilitate biogenesis of the eukaryotic initiation factor 2 (eIF2). J. Biol. Chem. 288, 21537−21546.
G
DOI: 10.1021/acssynbio.7b00372 ACS Synth. Biol. XXXX, XXX, XXX−XXX
