Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
30 Oct 2024
30 Oct 2024
Historique:
received:
01
12
2023
accepted:
23
10
2024
medline:
31
10
2024
pubmed:
31
10
2024
entrez:
31
10
2024
Statut:
epublish
Résumé
The development of artificial cells has led to fundamental insights into the functional processes of living cells while simultaneously paving the way for transformative applications in biotechnology and medicine. A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We modify the membrane translocating loop of α-hemolysin to translocate functional peptides up to 52 amino acids across lipid membranes. Full membrane translocation occurs in the absence of any translocase machinery and the translocated peptides are recognized by specific peptide-binding ligands on the opposing membrane side. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures.
Identifiants
pubmed: 39477950
doi: 10.1038/s41467-024-53783-4
pii: 10.1038/s41467-024-53783-4
doi:
Substances chimiques
Hemolysin Proteins
0
Peptides
0
Membranes, Artificial
0
Escherichia coli Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
9363Subventions
Organisme : U.S. Department of Defense (United States Department of Defense)
ID : N00014-22-1-2800
Informations de copyright
© 2024. The Author(s).
Références
Edidin, M. Lipids on the frontier: a century of cell-membrane bilayers. Nat. Rev. Mol. Cell. Biol. 4, 414–418 (2003).
pubmed: 12728275
doi: 10.1038/nrm1102
Cymer, F., von Heijne, G. & White, S. H. Mechanisms of integral membrane protein insertion and folding. J. Mol. Biol. 427, 999–1022 (2015).
pubmed: 25277655
doi: 10.1016/j.jmb.2014.09.014
Osborne, A. R., Rapoport, T. A. & van den Berg, B. Protein translocation by the Sec61/Secy channel. Annu. Rev. Cell Dev. 21, 529–550 (2005).
doi: 10.1146/annurev.cellbio.21.012704.133214
Guindani, C., da Silva, L. C., Cao, S., Ivanov, T. & Landfester, K. Synthetic cells: From simple bio-inspired modules to sophisticated integrated systems. Angew. Chem. Int. Ed. 61, e202110855 (2022).
doi: 10.1002/anie.202110855
Matsubayashi, H., Kuruma, Y. & Ueda, T. In vitro synthesis of the E. coli Sec translocon from DNA. Angew. Chem. Int. Ed. 53, 7535–7538 (2014).
doi: 10.1002/anie.201403929
Noba, K. et al. Bottom-up creation of an artificial cell covered with the adhesive bacterionanofiber protein AtaA. J. Am. Chem. Soc. 141, 19058–19066 (2019).
pubmed: 31697479
doi: 10.1021/jacs.9b09340
Manzer, Z. et al. Cell-free synthesis of a transmembrane mechanosensitive channel protein into a hybrid-supported lipid bilayer. ACS Appl. Bio Mater. 4, 3101–3112 (2021).
pubmed: 35014398
doi: 10.1021/acsabm.0c01482
Robelek, R. et al. Incorporation of in vitro synthesized GPCR into a tethered artificial lipid membrane system. Angew. Chem. Int. Ed. 46, 605–608 (2007).
doi: 10.1002/anie.200602231
Yabal, M. et al. Translocation of the C terminus of a tail-anchored protein across the endoplasmic reticulum membrane in yeast mutants defective in signal peptide-driven translocation. J. Biol. Chem. 278, 3489–3496 (2003).
pubmed: 12446686
doi: 10.1074/jbc.M210253200
Brambillasca, S., Yabal, M., Makarow, M. & Borgese, N. Unassisted translocation of large polypeptide domains across phospholipid bilayers. J. Cell Biol. 175, 767–777 (2006).
pubmed: 17130291
pmcid: 2064676
doi: 10.1083/jcb.200608101
Corcoran, J. A. et al. Myristoylation, a protruding loop, and structural plasticity are essential features of a nonenveloped virus fusion peptide motif. J. Biol. Chem. 279, 51386–51394 (2004).
pubmed: 15448165
doi: 10.1074/jbc.M406990200
Chang, D. K., Cheng, S. F. & Chien, W. J. The amino-terminal fusion domain peptide of human immunodeficiency virus type 1 gp41 inserts into the sodium dodecyl sulfate micelle primarily as a helix with a conserved glycine at the micelle-water interface. J. Virol. 71, 6593–6602 (1997).
pubmed: 9261381
pmcid: 191937
doi: 10.1128/jvi.71.9.6593-6602.1997
Islam, Md. Z. & Sharmin, S. Moniruzzaman, Md. & Yamazaki, M. Elementary processes for the entry of cell-penetrating peptides into lipid bilayer vesicles and bacterial cells. Appl. Microbiol. Biotechnol. 102, 3879–3892 (2018).
pubmed: 29523934
doi: 10.1007/s00253-018-8889-5
Gilbert, R. J. C. Pore-forming toxins. Cell. Mol. Life Sci. 59, 832–844 (2002).
pubmed: 12088283
pmcid: 11146115
doi: 10.1007/s00018-002-8471-1
Dinges, M. M., Orwin, P. M. & Schlievert, P. M. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13, 16–34 (2000).
pubmed: 10627489
pmcid: 88931
doi: 10.1128/CMR.13.1.16
Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. USA 101, 17669–17674 (2004).
pubmed: 15591347
pmcid: 539773
doi: 10.1073/pnas.0408236101
Deamer, D., Akeson, M. & Branton, D. Three decades of nanopore sequencing. Nat. Biotechnol. 34, 518–524 (2016).
pubmed: 27153285
pmcid: 6733523
doi: 10.1038/nbt.3423
Walker, B. & Bayley, H. Key residues for membrane binding, oligomerization, and pore forming activity of Staphylococcal α-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. J. Biol. Chem. 270, 23065–23071 (1995).
pubmed: 7559447
doi: 10.1074/jbc.270.39.23065
Palmer, M. et al. Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186. J. Biol. Chem. 268, 11959–11962 (1993).
pubmed: 8505320
doi: 10.1016/S0021-9258(19)50293-9
Valeva, A. et al. 18 Transmembrane β-barrel of staphylococcal α-toxin forms in sensitive but not in resistant cells. Proc. Natl. Acad. Sci. USA 94, 11607–11611 (1997).
pubmed: 9326657
pmcid: 23553
doi: 10.1073/pnas.94.21.11607
Ward, R. J., Palmer, M., Leonard, K. & Bhakdi, S. Identification of a putative membrane-inserted segment in the alpha-toxin of Staphylococcus aureus. Biochemistry 33, 7477–7484 (1994).
pubmed: 8003513
doi: 10.1021/bi00189a056
Krasilnikov, O. V., Capistrano, M.-F. P., Yuldasheva, L. N. & Nogueira, R. A. Influence of cys-130 S. aureus alpha-toxin on planar lipid bilayer and erythrocyte membranes. J. Membr. Biol. 156, 157–172 (1997).
pubmed: 9075647
doi: 10.1007/s002329900198
Krishnasastry, M., Walker, B., Braha, O. & Bayley, H. Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal α-hemolysin. FEBS Lett. 356, 66–71 (1994).
pubmed: 7988723
doi: 10.1016/0014-5793(94)01240-7
Walker, B., Kasianowicz, J., Krishnasastry, M. & Bayley, H. A pore-forming protein with a metal-actuated switch. Protein Eng. Des. Sel. 7, 655–662 (1994).
doi: 10.1093/protein/7.5.655
Russo, M. J., Bayley, H. & Toner, M. Reversible permeabilization of plasma membranes with an engineered switchable pore. Nat. Biotechnol. 15, 278–282 (1997).
pubmed: 9062930
doi: 10.1038/nbt0397-278
Hall, A. R. et al. Hybrid pore formation by directed insertion of α-haemolysin into solid-state nanopores. Nat. Nanotechnol. 5, 874–877 (2010).
pubmed: 21113160
pmcid: 3137937
doi: 10.1038/nnano.2010.237
Valeva, A. et al. Membrane insertion of the heptameric staphylococcal alpha-toxin pore. A domino-like structural transition that is allosterically modulated by the target cell membrane. J. Biol. Chem. 276, 14835–14841 (2001).
pubmed: 11279048
doi: 10.1074/jbc.M100301200
Harrington, L., Alexander, L. T., Knapp, S. & Bayley, H. Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor. Angew. Chem. Int. Ed. 54, 8154–8159 (2015).
doi: 10.1002/anie.201503141
Harrington, L., Cheley, S., Alexander, L. T., Knapp, S. & Bayley, H. Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. PNAS 110, E4417–E4426 (2013).
pubmed: 24194548
pmcid: 3839778
doi: 10.1073/pnas.1312739110
Pautot, S., Frisken, B. J. & Weitz, D. A. Production of unilamellar vesicles using an inverted emulsion. Langmuir 19, 2870–2879 (2003).
doi: 10.1021/la026100v
Zorzi, A., Deyle, K. & Heinis, C. Cyclic peptide therapeutics: past, present and future. Curr. Opin. Chem. Biol. 38, 24–29 (2017).
pubmed: 28249193
doi: 10.1016/j.cbpa.2017.02.006
de Herder, W. W. & Lamberts, S. W. J. Somatostatin and somatostatin analogues: diagnostic and therapeutic uses. Curr. Opin. Oncol. 14, 53 (2002).
pubmed: 11790981
doi: 10.1097/00001622-200201000-00010
Müller, T. D. et al. Glucagon-like peptide 1 (GLP-1). Mol. Metab. 30, 72–130 (2019).
pubmed: 31767182
pmcid: 6812410
doi: 10.1016/j.molmet.2019.09.010
Fujii, S., Matsuura, T. & Yomo, T. Membrane curvature affects the formation of α-hemolysin nanopores. ACS Chem. Biol. 10, 1694–1701 (2015).
pubmed: 25860290
doi: 10.1021/acschembio.5b00107
Pang, S. S. et al. The cryo-EM structure of the acid activatable pore-forming immune effector macrophage-expressed gene 1. Nat. Commun. 10, 4288 (2019).
pubmed: 31537793
pmcid: 6753088
doi: 10.1038/s41467-019-12279-2
Aksoyoglu, M. A. et al. Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin. Proc. Natl. Acad. Sci. USA 113, 9003–9008 (2016).
pubmed: 27466408
pmcid: 4987803
doi: 10.1073/pnas.1602716113
Machado, D. C. et al. Effects of alkali and ammonium ions in the detection of poly(ethyleneglycol) by alpha-hemolysin nanopore sensor. RSC Adv 6, 56647–56655 (2016).
doi: 10.1039/C6RA09234A
Jung, Y., Cheley, S., Braha, O. & Bayley, H. The internal cavity of the Staphylococcal α-hemolysin pore accommodates ∼175 exogenous amino acid residues. Biochemistry 44, 8919–8929 (2005).
pubmed: 15966717
doi: 10.1021/bi0473713
Nishimura, K. et al. Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry. Langmuir 28, 8426–8432 (2012).
pubmed: 22578080
doi: 10.1021/la3001703
Villar, G., Graham, A. D. & Bayley, H. A tissue-like printed material. Science 340, 48–52 (2013).
pubmed: 23559243
pmcid: 3750497
doi: 10.1126/science.1229495
Bayley, H., Cazimoglu, I. & Hoskin, C. E. G. Synthetic tissues. Emerg. Top. Life Sci. 3, 615–622 (2019).
pubmed: 33523175
pmcid: 7289033
doi: 10.1042/ETLS20190120
Booth, M. J., Schild, V. R., Graham, A. D., Olof, S. N. & Bayley, H. Light-activated communication in synthetic tissues. Sci. Adv. 2, e1600056 (2016).
pubmed: 27051884
pmcid: 4820383
doi: 10.1126/sciadv.1600056
Chiruvolu, S. et al. Higher order self-assembly of vesicles by site-specific binding. Science 264, 1753–1756 (1994).
pubmed: 8209255
doi: 10.1126/science.8209255
Carrara, P., Stano, P. & Luisi, P. L. Giant vesicles “colonies”: a model for primitive cell communities. ChemBioChem 13, 1497–1502 (2012).
pubmed: 22689306
doi: 10.1002/cbic.201200133
Hadorn, M., Boenzli, E. & Hanczyc, M. M. Specific and Reversible DNA-Directed Self-Assembly of Modular Vesicle-Droplet Hybrid Materials. Langmuir 32, 3561–3566 (2016).
pubmed: 27010467
doi: 10.1021/acs.langmuir.5b04003
Belardi, B. et al. Claudin-4 reconstituted in unilamellar vesicles is sufficient to form tight interfaces that partition membrane proteins. J. Cell Sci. 132, jcs221556 (2018).
pubmed: 30209136
pmcid: 6398474
doi: 10.1242/jcs.221556
van Roy, F. & Berx, G. The cell-cell adhesion molecule E-cadherin. Cell. Mol. Life Sci. 65, 3756–3788 (2008).
pubmed: 18726070
pmcid: 11131785
doi: 10.1007/s00018-008-8281-1
Wörsdörfer, B., Woycechowsky, K. J. & Hilvert, D. Directed evolution of a protein container. Science 331, 589–592 (2011).
pubmed: 21292977
doi: 10.1126/science.1199081
Beck, T., Tetter, S., Künzle, M. & Hilvert, D. Construction of Matryoshka-type structures from supercharged protein nanocages. Angew. Chem. Int. Ed. 54, 937–940 (2015).
doi: 10.1002/anie.201408677
Sun, H. et al. Self-assembly of cricoid proteins induced by “soft nanoparticles”: an approach to design multienzyme-cooperative antioxidative systems. ACS Nano 9, 5461–5469 (2015).
pubmed: 25952366
doi: 10.1021/acsnano.5b01311
Lin, A. J., Sihorwala, A. Z. & Belardi, B. Engineering tissue-scale properties with synthetic cells: Forging one from many. ACS Synth. Biol. 12, 1889–1907 (2023).
pubmed: 37417657
pmcid: 11017731
doi: 10.1021/acssynbio.3c00061
Mantri, S., Tanuj Sapra, K., Cheley, S., Sharp, T. H. & Bayley, H. An engineered dimeric protein pore that spans adjacent lipid bilayers. Nat. Commun. 4, 1725 (2013).
pubmed: 23591892
doi: 10.1038/ncomms2726
Abuin, E., Lissi, E. & Ahumada, M. Diffusion of hydrogen peroxide across DPPC large unilamellar liposomes. Chem. Phys. Lipids 165, 656–661 (2012).
pubmed: 22796350
doi: 10.1016/j.chemphyslip.2012.07.001
Pak, V. V. et al. Ultrasensitive genetically encoded indicator for hydrogen peroxide identifies roles for the oxidant in cell migration and mitochondrial function. Cell Metab. 31, 642–653.e6 (2020).
pubmed: 32130885
pmcid: 7088435
doi: 10.1016/j.cmet.2020.02.003
Wang, X. et al. Chemical information exchange in organized protocells and natural cell assemblies with controllable spatial positions. Small 16, 1906394 (2020).
doi: 10.1002/smll.201906394
Li, S., Wang, X., Mu, W. & Han, X. Chemical signal communication between two protoorganelles in a lipid-based artificial cell. Anal. Chem. 91, 6859–6864 (2019).
pubmed: 31020837
doi: 10.1021/acs.analchem.9b01128
Yang, B., Li, S., Mu, W., Wang, Z. & Han, X. Light-harvesting artificial cells containing cyanobacteria for CO2 fixation and further metabolism mimicking. Small 19, 2201305 (2023).
doi: 10.1002/smll.202201305