Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation

Nature Neuroscience volume 17, pages 471–480 (2014)Cite this article

Subjects

Abstract

Rapid and reversible methods for altering the levels of endogenous proteins are critically important for studying biological systems and developing therapeutics. Here we describe a membrane-permeant targeting peptide–based method that rapidly and reversibly knocks down endogenous proteins through chaperone-mediated autophagy in vitro and in vivo. We demonstrate the specificity, efficacy and generalizability of the method by showing efficient knockdown of various proteins, including death associated protein kinase 1 (160 kDa), scaffolding protein PSD-95 (95 kDa) and α-synuclein (18 kDa), with their respective targeting peptides in a dose-, time- and lysosomal activity–dependent manner in rat neuronal cultures. Moreover, we show that, when given systemically, the peptide system efficiently knocked down the targeted protein in the brains of intact rats. Our study provides a robust and convenient research tool for manipulating endogenous protein levels and may also lead to the development of protein knockdown–based therapeutics for treating human diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: CTM-directed protein degradation.
Figure 2: DAPK1 targeting peptide knocks down active DAPK1 in HEK cells.
Figure 3: DAPK1-targeting peptide specifically degrades activated endogenous DAPK1 in neuronal culture.
Figure 4: Target peptide–mediated respective degradation of α-synuclein and PSD-95 in cultured neurons.
Figure 5: TAT-GluN2Bct-CTM knocks down H2O2-activated DAPK1, protecting neurons against H2O2-induced neurotoxicity in neuronal cultures.
Figure 6: TAT-GluN2BCTM specifically knocks down DAPK1 in ischemic brain areas and reduces neuronal damage in the MCAo model of focal ischemia in rats.

Similar content being viewed by others

References

  1. Houdebine, L.-M. Transgenic animal models in biomedical research. Methods Mol. Biol. 360, 163–202 (2007).

    CAS  PubMed  Google Scholar 

  2. Yamamoto, A., Hen, R. & Dauer, W.T. The ons and offs of inducible transgenic technology: a review. Neurobiol. Dis. 8, 923–932 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Kole, R., Krainer, A.R. & Altman, S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat. Rev. Drug Discov. 11, 125–140 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Castanotto, D. & Rossi, J.J. The promises and pitfalls of RNA-interference-based therapeutics. Nature 457, 426–433 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Banaszynski, L.A. & Wandless, T.J. Conditional control of protein function. Chem. Biol. 13, 11–21 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Caussinus, E., Kanca, O. & Affolter, M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat. Struct. Mol. Biol. 19, 117–121 (2011).

    Article  PubMed  Google Scholar 

  7. Bonger, K.M., Chen, L.-C., Liu, C.W. & Wandless, T.J. Small-molecule displacement of a cryptic degron causes conditional protein degradation. Nat. Chem. Biol. 7, 531–537 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Neklesa, T.K. et al. Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7, 538–543 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Banaszynski, L.A., Chen, L.-C., Maynard-Smith, L.A., Ooi, A.G.L. & Wandless, T.J.A. Rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Sakamoto, K.M. et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98, 8554–8559 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dice, J.F. Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem. Sci. 15, 305–309 (1990).

    Article  CAS  PubMed  Google Scholar 

  13. Kaushik, S. & Cuervo, A.M. Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol. 22, 407–417 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Koga, H., Martinez-Vicente, M., Macian, F., Verkhusha, V.V. & Cuervo, A.M. A photoconvertible fluorescent reporter to track chaperone-mediated autophagy. Nat. Commun. 2, 386 (2011).

    Article  PubMed  Google Scholar 

  15. Backer, J.M., Bourret, L. & Dice, J.F. Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids. Proc. Natl. Acad. Sci. USA 80, 2166–2170 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cuervo, A.M. & Dice, J.F. Unique properties of lamp2a compared to other lamp2 isoforms. J. Cell Sci. 113, 4441–4450 (2000).

    CAS  PubMed  Google Scholar 

  17. Slot, L.A., Lauridsen, A.-M. & Hendil, K. Intracellular protein degradation in serum-deprived human fibroblasts. Biochem. J. 237, 491–498 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Seglen, P.O. & Reith, A. Ammonia inhibition of protein degradation in isolated rat hepatocytes. Quantitative ultrastructural alterations in the lysosomal system. Exp. Cell Res. 100, 276–280 (1976).

    Article  CAS  PubMed  Google Scholar 

  19. Bauer, P.O. et al. Harnessing chaperone-mediated autophagy for the selective degradation of mutant huntingtin protein. Nat. Biotechnol. 28, 256–263 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Cataldo, A.M. & Nixon, R.A. Enzymatically active lysosomal proteases are associated with amyloid deposits in Alzheimer brain. Proc. Natl. Acad. Sci. USA 87, 3861–3865 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Neff, N.T., Bourret, L., Miao, P. & Dice, J.F. Degradation of proteins microinjected into IMR-90 human diploid fibroblasts. J. Cell Biol. 91, 184–194 (1981).

    Article  CAS  PubMed  Google Scholar 

  22. Cuervo, A.M. Impaired degradation of mutant -synuclein by chaperone-mediated autophagy. Science 305, 1292–1295 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Cuervo, A.M., Terlecky, S.R., Dice, J.F. & Knecht, E. Selective binding and uptake of ribonuclease A and glyceraldehyde-3-phosphate dehydrogenase by isolated rat liver lysosomes. J. Biol. Chem. 269, 26374–26380 (1994).

    CAS  PubMed  Google Scholar 

  24. Henshall, D.C. et al. Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures. J. Neurochem. 86, 1260–1270 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Shamloo, M. et al. Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia. J. Biol. Chem. 280, 42290–42299 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Tu, W. et al. DAPK1 interaction with NMDA receptor GluN2B subunits mediates brain damage in stroke. Cell 140, 222–234 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cohen, O., Feinstein, E. & Kimchi, A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J. 16, 998–1008 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Vivès, E., Brodin, P. & Lebleu, B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010–16017 (1997).

    Article  PubMed  Google Scholar 

  29. Traynelis, S.F. et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405–496 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Morris, M.C., Depollier, J., Mery, J., Heitz, F. & Divita, G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173–1176 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Wang, Y. α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptor (AMPAR) endocytosis is essential for N-methyl-D-aspartate-induced neuronal apoptosis. J. Biol. Chem. 279, 41267–41270 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Spillantini, M.G. et al. α-Synuclein in Lewy bodies. Nature 388, 839–840 (1997).

    Article  CAS  PubMed  Google Scholar 

  33. Shaltiel-Karyo, R. et al. Inhibiting α-synuclein oligomerization by stable cell-penetrating β-synuclein fragments recovers phenotype of Parkinson's disease model flies. PLoS ONE 5, e13863 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Aarts, M. et al. Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions. Science 298, 846–850 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Iadecola, C. & Anrather, J. Stroke research at a crossroad: asking the brain for directions. Nat. Neurosci. 14, 1363–1368 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Eisenberg-Lerner, A. & Kimchi, A. DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress. Cell Death Differ. 14, 1908–1915 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Ricart, K.C. & Fiszman, M.L. Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media. Neurochem. Res. 26, 801–808 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Liu, F., Schafer, D.P. & McCullough, L.D. TTC, fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion. J. Neurosci. Methods 179, 1–8 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Taghibiglou, C. et al. Role of NMDA receptor–dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries. Nat. Med. 15, 1399–1406 (2009).

    Article  CAS  PubMed  Google Scholar 

  40. Heitz, F., Morris, M.C. & Divita, G. Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br. J. Pharmacol. 157, 195–206 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Tymianski, M. Can molecular and cellular neuroprotection be translated into therapies for patients?: yes, but not the way we tried it before. Stroke 41, S87–S90 (2010).

    Article  PubMed  Google Scholar 

  42. Banaszynski, L.A., Sellmyer, M.A., Contag, C.H., Wandless, T.J. & Thorne, S.H. Chemical control of protein stability and function in living mice. Nat. Med. 14, 1123–1127 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Mason, J.M. Design and development of peptides and peptide mimetics as antagonists for therapeutic intervention. Future Med. Chem. 2, 1813–1822 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Stein, A. & Aloy, P. Contextual specificity in peptide-mediated protein interactions. PLoS ONE 3, e2524 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Cuervo, A.M. Chaperone-mediated autophagy: selectivity pays off. Trends Endocrinol. Metab. 21, 142–150 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Hill, M.D. et al. Safety and efficacy of NA-1 in patients with iatrogenic stroke after endovascular aneurysm repair (ENACT): a phase 2, randomised, double-blind, placebo-controlled trial. Lancet Neurol. 11, 942–950 (2012).

    Article  CAS  PubMed  Google Scholar 

  47. Foy, K.C., Liu, Z., Phillips, G., Miller, M. & Kaumaya, P.T.P. Combination treatment with HER-2 and VEGF peptide mimics induces potent anti-tumor and anti-angiogenic responses in vitro and in vivo. J. Biol. Chem. 286, 13626–13637 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Bartlett, T. et al. Slice orientation and muscarinic acetylcholine receptor activation determine the involvement of N-methyl d-aspartate receptor subunit GluN2B in hippocampal area CA1 long-term depression. Mol. Brain. 4, 41 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Liu, Y. et al. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J. Neurosci. 27, 2846–2857 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Peineau, S. et al. LTP inhibits LTD in the hippocampus via regulation of GSK3β. Neuron 53, 703–717 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Taghibiglou, C., Lu, J., Mackenzie, I.R., Wang, Y.T. & Cashman, N.R. Sterol regulatory element binding protein-1 (SREBP1) activation in motor neurons in excitotoxicity and amyotrophic lateral sclerosis (ALS): Indip, a potential therapeutic peptide. Biochem. Biophys. Res. Commun. 413, 159–163 (2011).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Y. Li for technical support and L. Oschipok for editorial assistance. We also thank L. Luo for help in obtaining the whole-brain images of DAPK1 immunostaining (Fig. 6f). This work was supported by the Canadian Institutes of Health Research, Heart and Stroke Foundation of British Columbia and Yukon, and Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004). This research is also in part supported by a research grant from Brain Canada, Genome British Columbia and the Michael Smith Foundation for Health Research.

Author information

Author notes

  1. Xuelai Fan and Wu Yang Jin: These authors contributed equally to this work.

Authors and Affiliations

  1. Brain Research Centre and Department of Medicine, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, Canada

    Xuelai Fan, Wu Yang Jin, Jie Lu & Yu Tian Wang

  2. Institute of Pharmacology, Medicine College of Shandong University, Jinan, China

    Jin Wang

  3. Translational Medicine Research Center, China Medical University Hospital, Taichung, Taiwan

    Yu Tian Wang

  4. Graduate Institute of Immunology, China Medical University, Taichung, Taiwan

    Yu Tian Wang

Authors
  1. Xuelai Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wu Yang Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jie Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yu Tian Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.F. and W.Y.J. designed and performed experiments, as well as analyzed the data. X.F. also wrote the manuscript. J.L. and J.W. assisted in performing some of the molecular biochemical experiments. Y.T.W. designed the study, supervised the overall project and wrote the manuscript.

Corresponding author

Correspondence to Yu Tian Wang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Design of the CMA targeting peptide and time-dependent degradation by CMA.

(a) Schematic diagram illustrating the design of the targeting peptide mediated protein degradation. Following administration, the targeting peptide enters the cell through its cell membrane penetrating domain (CMPD), binds to the target protein via its protein binding domain (PBD), and chaperones the peptide-protein complex to the lysosome for degradation via its chaperone-mediated autophagy targeting motif (CTM). (b) CTM-GFP is degraded in a time-dependent manner, without affecting the stability of endogenous CMA-substrate GAPDH in HEK cells transiently transfected with CTM-GFP. N=3 from 3 separate cultures and transfections. One-way ANOVA F(9,20)=12.418, p<0.001. Bars represent protein levels normalized to 12h WT-GFP (white bar), and compared to 12h WT-GFP (white bar, *) or 12h CTM-GFP (grey bar, Δ) *,Δ p<0.05, **,ΔΔ p<0.01 and ***, ΔΔΔp<0.001, bars represent relative mean values±s.e.m. Full-length blots are available in Supplementary Figure 9.

Supplementary Figure 2 TAT-GluN2BCTM does not alter Dapk1 mRNA levels.

Cultured cortical neurons were pre-treated with TAT-GluN2BCTM (25μM, 60min prior to and during NMDA application) and NMDA (50μM;30min). 2hrs (n=5), 4hrs (n=4) and 7hrs (n=4) following NMDA washout, total RNA was extracted and reverse-transcribed into cDNA. Dapk1 mRNA levels were measured by q-PCR with β-actin (Actb) used as an internal control. F(5,25)=0.134, p=0.983 One-way ANOVA. Bars represent relative mean values±s.e.m. mRNA was collected from primary cells from at least 3 separate cultures.

Supplementary Figure 3 TAT-GluN2BCTM–induced degradation of DAPK1 reduces DAPK1 levels in various subcellular compartments in neuron cultures.

Bath applications of TAT-GluN2BCTM (25μM, 1h prior to and during NMDA treatment) significantly decreased DAPK1 in nuclear (left, n=4, p<0.001, F(2,9)=19.139), cytosolic (middle, n=4, p=0.006, F(2,8)=10.417) and mitochondrial (right, n=4, p<0.001, F(2,9)=18.597) subcellular fractions 2h after NMDA treatment and washout, as compared to saline control and NMDA-treated group (grey bar). Cells were collected from at least 3 separate primary cultures. Lysates were collected as a mixture of all treatment samples. One-way ANOVA with Tukey post hoc. * compared to control, Δ compared to NMDA-treated group (grey bar). *p<0.05, ΔΔp<0.01; ***, ΔΔp<0.001bars represent relative mean values±s.e.m. normalized to saline control (arbitrarily set as 1). Full-length blots are available in Supplementary Figure 9.

Supplementary Figure 4 siRNA-directed knockdown of Lamp2a reduces TAT-GluN2BCTM induced DAPK1 degradation.

(a) DNA electrophoresis of Lamp2a showing siRNA (60pmol)-directed knockdown of Lamp2a 3d after siRNA treatment. Scrb-siRNA: scrambled siRNA, LAMP-2A-siRNA: Lamp2a-targeting siRNA. N=3. (b-c) Sequential immunoblotting showing the inhibition of TAT-GluN2BCTM induced knockdown of DAPK1 by specifically blocking CMA with siRNA-mediated Lamp2a knockdown. 3d following siRNA treatment, cells were incubated in TAT-GluN2BCTM (25μM) for 1hr before and during NMDA (50μM; 30min). Cells were harvested 2h after the peptide and NMDA washout. β-actin was used as loading control. Lamp2aknockdown: n=7, One-way ANOVA p<0.001, F(3,24)=13.455; DAPK1 Knockdown: n=7, Kruskal-Wallis One-Way ANOVA on Ranks with Tukey post hoc, p=0.002, H(3)=15.047; * compared to saline control, Δ compared to NMDA-treated group (grey bar). *,Δ p<0.05, ***p<0.001; bars represent relative mean values±s.e.m. normalized to saline control (arbitrarily set as 1).Cells were collected from at least 3 separate primary cultures. Full-length blots are available in Supplementary Figure 9.

Supplementary Figure 5 Pep1-mediated intracellular delivery of short synthetic GluN2B-CTM peptides specifically knocks down active native DAPK1 in cultured neurons.

(a) Schematic illustration of synthetic peptide GluN2B-CTM. (b) GluN2B-CTM, when delivered into cortical neurons, specifically decreases the level of native DAPK1 in an NMDA-stimulation dependent manner. GluN2B-CTM was first mixed with intracellular delivering carrier peptide Pep-1 at a 1:4 ratio for 30min to form a plasma membrane permeable peptide complex, which was then bath applied to neurons 60min prior to and during NMDA treatments (50μM; 30min). GluN2B-CTM plus Pep-1 (but not the Pep-1 alone) dose-dependently (b, p<0.001 F(7,22)=6.993, from at least 2 individual experiments) and time-dependently (c, p<0.001, F(7,28)=10.034, from at least 3 individual experiments) decreased the level of endogenous DAPK1 in cultured cortical neurons following NMDA treatment. The reduction required NMDA stimulation (b) and was rescued by inhibiting lysosome function with NH4Cl (d; NH4Cl 20mM; n=5; One-way ANOVA, p<0.001, F(3,16)=15.129.). Bars represent relative DAPK1 levels normalized to saline group (white bar, arbitrarily set as 1), and compared to both saline (white bar, *) or NMDA-treated group (grey bar, Δ). Membrane re-probing for β-actin was used as loading control. *,Δ p<0.05, **,ΔΔ p<0.01 and ***, ΔΔΔp<0.001; bars represent relative mean values±s.e.m. normalized to the saline control (white bar, arbitrarily set as 1). Cells were collected from at least 3 separate primary cultures. Full-length blots are available in Supplementary Figure 9.

Supplementary Figure 6 Immunoblots showing that the macroautophagy inhibitor 3-methyladenine (3-MA) cannot rescue targeting-peptide mediated degradation.

(a) α-synuclein targeting peptide TAT-βsynCTM (25μM) significantly decreased native α-synuclein in the presence of 3-methyladenine (10mM). Basal level of α-synuclein showed a non-significant trend of increase with the addition of 3-methyladenine. n=5, p<0.001, F(3,16)=47.013. (b) 3-methyladenine did not alter either the basal levels or NMDA activation-dependent knockdown of DAPK1 by its targeting peptide TAT-GluN2BCTM. n=4, p<0.001, F(3,12)=21.675. (c) Similarly, 3-methyladenine failed to affect either the basal or the TAT-GluN2B9cCTM reduced level of PSD-95. n=4, p<0.001, F(3,12)=14.836. β-actin was used as loading control. One-way ANOVA with Tukey post hoc. * p<0.05, **p<0.01 and ***p<0.001; bars represent relative mean values±s.e.m. normalized to saline control (arbitrarily set as 1). Cells were collected from at least 3 separate primary cultures. Full-length blots are available in Supplementary Figure 9.

Supplementary Figure 7 α-synuclein(ΔDQ) and α-synuclein A53T are degraded by TAT-βsynCTM.

The endogenous α-synuclein CMA targeting motif was eliminated by mutating 95VKKDQ99 into 95VKKAA99. Plasmids expressing wild-type α-synuclein, α-synuclein (ΔDQ) and α-synuclein A53T were transfected into HEK293 cells and treated with either TAT-βsyn (50μM) or TAT-βsynCTM (50μM) starting at 24h post-transfection. Two additional doses of peptide were added 4 and 8h later, and cells were harvested at 48h after transfection. Wild-type α-synuclein was significantly degraded by TAT-βsynCTM, which was rescued by inhibiting the lysosome with NH4Cl (n=4, H(2)=8.290, p=0.005, Kruskal-Wallis One-way ANOVA on Ranks). α-synuclein(ΔDQ) was degraded by TAT-βsynCTM (n=4, F(2,11)=9.482, p=0.006, One-way ANOVA), which was rescued by NH4Cl (n=4, p=0.001). Similarily, α-synuclein A53T was degraded by the targeting peptide (n=4, F(2,11)=6.340, p=0.019, One-way ANOVA), which was rescued by NH4Cl (n=4, p=0.019). Student-Newman-Keuls was used for post-hoc analysis. β-actin was used as loading control. * compared to TAT-βsyn, Δ compared to TAT- βsynCTM *,Δ p<0.05, **p<0.01; bars represent relative mean values±s.e.m. Cells were collected from at least 4 separate cultures and transfections. Full-length blots are available in Supplementary Figure 9.

Supplementary Figure 8 Targeting peptides do not show significant cell toxicity 24 h after treatments.

Cortical neurons were treated with 25μM of recombinant (left, n=4, p=0.005 H(3)=12.706) or synthetic (right, n=4, p<0.001, H(7)=28.074) peptides, and 24h later, cell death was assessed by lactate dehydrogenase (LDH) assay. For positive control, cells were lysed with Triton-X 100 prior assay per the manufacture's instruction. Kruskal-Wallis One-Way ANOVA on Ranks was used for analysis. *p<0.05. Bars represent relative mean values±s.e.m. normalized to the saline control (arbitrarily set as 1). n=4 individual experiments from at least 2 separate primary cultures.

Supplementary Figure 9 Full-length immunoblots for individual figures.

(a) Fig. 1c; (b) Figs. 2b-e, note that membranes were cut at 70kDa and 35kDa for immunoblot; (c) Fig. 3a, Figs. 3c-f and Fig.3h, note in Fig. 3f s=single, m=multiple; (d) Fig.4a and 4b; (e) Fig. 5a and 5b, note in Fig.5a c=control; (f) Fig. 6d, note that membranes were cut around 170kDa before immunoblotting to facilitate viewing of DAPK1 band (160kDa); (g) Supplementary Fig. 1b; (h) Supplementary Fig. 3; (i) Supplementary Figs 4a-c; (j) Supplementary Figs. 5a-d; (k) Supplementary Figs. 6a-c; and (l) Suppl. Fig. 7.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 13361 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fan, X., Jin, W., Lu, J. et al. Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation. Nat Neurosci 17, 471–480 (2014). https://doi.org/10.1038/nn.3637

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3637

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing