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

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

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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.

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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.

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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.

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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

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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.

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Correspondence to Yu Tian Wang.

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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.

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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

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