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Designed proteins induce the formation of nanocage-containing extracellular vesicles

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Abstract

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1,2 or proteins3,4,5, but strategies for engineering hybrid biological materials are only beginning to emerge6,7. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as ‘enveloped protein nanocages’ (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (~100 nm) vesicles containing multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.

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Figure 1: EPN release requires three functional elements.
Figure 2: EPNs comprise cell-derived membrane envelopes containing multiple protein nanocages.
Figure 3: EPNs can package macromolecular cargoes and deliver them into new target cells.
Figure 4: A variety of functional elements and protein architectures support EPN formation.

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  • 07 December 2016

    Minor corrections were made to Fig. 4c and the legend of Fig. 3a.

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Acknowledgements

This work was supported in part by Deutsche Forschungsgemeinschaft (DFG) Fellowship VO 1836/1-1 (to J.V.), an NIH Molecular Biology Training Grant (T32GM008268) (Y.H.), a PHS National Research Service Award (T32GM007270) from NIGMS (U.N.), grants from the Bill & Melinda Gates Foundation (OPP1118840) and Defense Advanced Research Projects Agency (W911NF-14-1-0162 and W911NF-15-1-0645) (N.P.K.), and NIH grants RO1 AI 51174 and P50 082545 (W.I.S.). We thank P. Shen and J. McCullough for assistance and advice on cryo-EM experiments, M. Kay and D. Eckert and J. Marvin (University of Utah FACS Core) for help with BlaM assays, S. Carter and S. Magesrawan (Caltech) for assistance with Amira Software, L. Nikolova (University of Utah EM Core) for assistance with immunogold labelling experiments, M. Redd (University of Utah Cell Imaging Core) for assistance with confocal microscopy, A. Wargacki for cloning assistance, S. Hauschka for tissue culture assistance, and M. Lajoie, L. Stewart and D. Baker for helpful discussions.

Author information

Authors and Affiliations

Authors

Contributions

J.V., N.P.K. and W.I.S. designed and coordinated the study. J.V. performed EPN and cellular protein release assays, IP assays, BlaM delivery assays, confocal fluorescence microscopy, and immunogold EM. J.V. and D.M.B. performed cryo-electron microscopy and tomography, including single-particle reconstruction. C.O. and S.Y. designed EPN constructs and produced, purified, and analysed EPN proteins expressed in E. coli. C.O. performed EPN release assays, protease protection assays, and aldolase assays. Y.H. performed aldolase assays and purified I3-01 from E. coli. U.N. performed negative stain EM on proteins purified from E. coli. J.V., C.O., D.M.B., N.P.K. and W.I.S. interpreted data. J.V., N.P.K. and W.I.S. wrote the manuscript.

Corresponding authors

Correspondence to Neil P. King or Wesley I. Sundquist.

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Competing interests

J.V., Y.H., N.P.K. and W.I.S. co-authored a US patent application (62/126,331) with claims relating to Enveloped Protein Nanocages through the CoMotion office at the University of Washington. The authors declare no other competing financial interests.

Additional information

Reviewer Information

Nature thanks O. Farokhzad, J. Hurley and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 EPN-01* is released from cells.

a, Schematic of the EPN-01* constructs. Note that the EPN-01* constructs shown in this figure are analogous to the EPN-01 constructs shown in Fig. 1 except that EPN-01* ran as a doublet because a methionine codon at I3-01 position 3 (isoleucine in EPN-01) served as a second translation initiation codon. b, Western blots showing functional and mutant EPN-01* proteins harvested from 293T cell culture supernatants (top panel). The middle panels are western blots showing cellular EPN-01* proteins in the Triton-insoluble fraction (stably bound to membranes) and the Triton-soluble fraction (cytoplasm and weakly bound to membranes), respectively. The bottom panel is a western blot showing a GAPDH loading control from the Triton-soluble fraction. Right, the percentage of each protein released into the supernatant is plotted (error bars show standard deviation from three technical repetitions). In summary, EPN-01 and EPN-01* were functionally equivalent and were employed interchangeably in our studies. For western blot source data, see Supplementary Fig. 1.

Extended Data Figure 2 EPN-01* forms nanocages that resemble those of the I3-01 scaffold.

a, Size-exclusion chromatograms (Superose 6 10/300 GL) of EPN-01*, EPN-01(ΔM), EPN-01(ΔL1 + ΔL2), I3-01, and 1wa3-wt purified from E. coli. 1wa3-wt is the wild-type trimeric aldolase from which I3-01 was derived9. All of the proteins eluted as nanocages except for 1wa3-wt, which eluted at the volume expected for the trimer. b, SDS–PAGE of the peak fraction from each of the chromatograms in a. Molecular weight marker positions are indicated in kilodaltons. c, Negative stain electron microscopy image of purified E. coli EPN-01*, showing a homogeneous field of particles (~25 nm diameter).

Extended Data Figure 3 EPN-01 constructs are released within membrane vesicles.

a, Detergent-dependent antibody accessibility of EPN-01 protein released from mammalian cells. Western blots of EPN-01 harvested from mammalian cell supernatants (top two blots) or non-enveloped EPN-01* nanocages purified from E. coli (bottom two blots) are shown. Samples were treated with 0.1% or 0.5% CHAPS, as indicated. Lane 1 shows input protein, and lanes 2–5 show bound and unbound fractions from immunoprecipitations with anti-Myc-agarose (lanes 2 and 3) or control anti-rabbit-IgG-agarose (lanes 4 and 5). b, Detergent-dependent aldolase substrate accessibility of EPN-01* protein released from mammalian cells. EPN-01* produced in 293F cells (top) or E. coli (bottom) were monitored for KPDG aldolase activity in the presence or absence of detergent. Note that the protein produced in mammalian cells only shows enzymatic activity in the presence of detergent, whereas the bacterially produced protein is equally active in both conditions. c, Size distribution of EPNs released into the culture supernatants of 293T cells expressing EPN-01*. n = 142 EPNs total; mean size, 107 ± 44. d, EPN-01* and associated cellular proteins are released from 293T cells. Western blots showing levels of I3-01–Myc, EPN-01*, ALIX and actin expressed in 293T cells (left) and released into the culture supernatant (right). e, Numbers of EPN-01* protein nanocages encapsulated within six different EPN-01* vesicles of varying sizes. Nanocage numbers were determined by visual counting of reconstructed EPNs from cryo-tomographic tilt series. An average EPN vesicle of diameter 110 nm has a volume of 7 × 105 nm3 and each nanocage has an estimated volume of approximately 8 × 103 nm3. A 110 nm diameter vesicle could therefore theoretically contain as many as 90 close-packed nanocages, but they actually contain, on average, around 14 nanocages.

Extended Data Figure 4 EPN-01* protein released from 293T cells assembles into nanocages that closely correspond to the designed I3-01 structure.

a, Cryo-EM image of extracellular EPN-01* nanocages released from vesicles by CHAPS detergent treatment. b, Cryo-EM 2D class averages showing the ten most prevalent classes, together with the numbers of particles in each class. c, Fourier shell correlations between the two half model charge density maps unmasked (blue curve) and masked (magenta curve). Orange curve shows Fourier shell correlations between the final charge density model and the I3-01 design model (with an additional residue built into the N terminus).

Extended Data Figure 5 Intracellular localization of EPN-01 and EPN-18.

a, Confocal fluorescence images of HeLa cells transfected with EPN-01 (left) and EPN-01(ΔM) (right) stained for Myc (green), DNA (blue) and actin (red). Note that EPN-01 is localized primarily in intracellular compartments and also at the plasma membrane (white arrow), whereas EPN-01(ΔM) is cytoplasmic. b, Confocal fluorescence images of HeLa cells transfected with EPN-18 (left) and EPN-18(ΔM) (right). Note that EPN-18 is predominantly localized at the plasma membrane (white arrow), whereas EPN-18(ΔM) concentrates at internal puncta.

Extended Data Figure 6 Intracellular localization and extracellular release of EPN-01.

a, Immunogold labelling of thin sections from 293T cells expressing EPN-01. Right image shows the boundaries of plasma membrane (cyan), cytosolic (black) and intracellular compartments (red) used for quantification (see d). b, Immunogold labelling of thin sections from 293T cells expressing EPN-01(ΔM). Right image shows the boundaries used for quantification, colour-coded as in a, except that intracellular compartment(s) could not be identified in this case because the EPN-01(ΔM) mutant does not localize there. c, Immunogold labelling of thin sections from 293T cells expressing EPN-01 showing budding and released EPNs. Right image shows the boundaries used for quantification as in a, plus boundaries to quantify gold particle densities in released EPNs (green). Image to the left shows an expanded view of two EPNs that appear to be budding from the plasma membrane. d, Image quantification confirmed that EPN-01 is enriched at the plasma membrane, within intracellular compartments, and in released EPNs. The P value corresponding to the difference between plasma membrane localization of EPN-01 and EPN-01(ΔM) is 7 × 10−5, indicated by the three asterisks (unpaired t-test, n = 11 images for EPN-01 and n = 9 images for EPN-01(ΔM)).

Extended Data Figure 7 EPNs can package biological cargoes and deliver them to the cytoplasm of target HeLa cells.

a, Schematic illustration showing the production, assembly, and release of EPNs incorporating BlaM–Vpr and VSV-G proteins (left), and detection of uptake and target cell membrane fusion using a BlaM colourimetric activity assay (right). b, Co-expression of VSV-G increases the number of vesicles that contain spikes, as evaluated by scoring >140 vesicles as either ‘containing’ or ‘not containing’ surface spikes (images like the one shown in the inset of Fig. 3b were scored independently by two different people, one blinded, and their counts were averaged). c, Western blots showing cellular expression and release of EPN-01 and Myc-tagged GFP constructs with or without fused Vpr (see Supplementary Table 3 for sequence information). Top blot shows released protein, middle and bottom blots show expression of Myc-tagged proteins and GAPDH in whole-cell lysates, respectively. Lane 1 shows co-expression of EPN-01 with Myc–GFP–Vpr, lane 2 shows co-expression of EPN-01 with Myc–GFP. d, Flow cytometric analyses of HeLa cells loaded with the fluorescent CCF2 β-lactamase substrate and incubated with increasing quantities of wild-type EPN-01*/VSV-G/BlaM–Vpr (top row), EPN-01*/VSV-G(P127D) mutant/BlaM–Vpr (middle row), and EPN-01*(LF45AA) mutant/VSV-G/BlaM–Vpr (bottom row).

Extended Data Figure 8 Aldolase and protease protection assays for EPNs with a variety of functional elements and protein architectures.

Schematic illustrations and analyses of the 16 EPN constructs that yielded robust EPN biogenesis are shown, as well as one negative control. Each panel shows the construct, a representative plot of aldolase activity in the presence (black line) and absence (grey line) of detergent, and a western blot analysis of the protease protection assay. Arrowheads next to each blot denote the full-length protein. Aldolase activity was monitored by disappearance of absorbance at 339 nm. a, Different membrane-binding elements support EPN formation. b, EPN-02, also referred to as EPN-01(ΔM), is a negative control construct in which the myristoylation site was inactivated by mutation. Both assays reveal that EPN-02 protein was not released from cells. c, EPN-51, which uses the designed 24-subunit protein assembly O3-33 as a self-assembly domain, forms an EPN with an intact membrane envelope. The aldolase assay was not included because O3-33 is not an aldolase. d, Different ESCRT-recruiting elements can support EPN formation. The asterisk next to the blot of EPN-36 signifies that the blot was overexposed: EPN-36 reproducibly yielded fainter bands on western blots than would be expected based on its aldolase activity and analyses of SDS–PAGE gels stained with Coomassie. e, Membrane-binding, self-assembly, and ESCRT-recruiting elements can function from different positions within EPN constructs. EPN-11 is a permutation of EPN-07, while EPN-23, EPN-24, and EPN-25 are permutations of EPN-18.

Supplementary information

Supplementary Information

This file contains legends for Supplementary Videos 1 and 2, Supplementary Tables 1-4 and additional references. (PDF 934 kb)

Supplementary Figures

This file contains the uncropped scans with size marker indications for Figures 1b, 2a, 3a, 4a,b,c,d and Extended Data Figures 1, 2b, 3a,d, 7a and 8 (PDF 908 kb)

Cryo-EM tomographic reconstruction of a released EPN

Shown is the raw charge density in different slices of the tomogram (first pass) and isosurface renderings (second pass) of individual protein nanocages (gold) and the limiting membrane of the EPN (green). (MP4 22821 kb)

Single particle cryo-EM reconstruction of released EPN-01* protein nanocage.

Shown are the raw charge density (grey, contoured at 4.5 σ) and a rigid body fitting of the I3-01 design model (dark green) plus one Nterminal amino acid (yellow) added to I3-01. (MP4 10366 kb)

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Votteler, J., Ogohara, C., Yi, S. et al. Designed proteins induce the formation of nanocage-containing extracellular vesicles. Nature 540, 292–295 (2016). https://doi.org/10.1038/nature20607

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