SNIPER Peptide‐Mediated Degradation of Endogenous Proteins

Xuelai Fan1, Yu Tian Wang2

1 Brain Research Centre and Department of Medicine, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, 2 Translational Medicine Research Center, China Medical University Hospital, Taichung
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140202
Online Posting Date:  March, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Rapid and reversible methods for altering the function of endogenous proteins are not only indispensable tools for probing complex biological systems, but may potentially drive the development of new therapeutics for the treatment of human diseases. Genetic approaches have provided insights into protein function, but are limited in speed, reversibility and spatiotemporal control. To overcome these limitations, we have developed a peptide‐based method (SNIPER: Selective Native Protein Eradication) to degrade any given endogenous protein at the post‐translational level by harnessing chaperone‐mediated autophagy, a major intracellular protein degradation pathway. This unit presents a typical strategy in the design and validation of a protein‐knockdown peptide. © 2015 by John Wiley & Sons, Inc.

Keywords: peptide; lysosome; protein knockdown; chaperone‐mediated autophagy

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of CPP‐Linked Protein‐Knockdown Peptides
  • Basic Protocol 2: Confirmation of Binding Between Peptide and Target Protein
  • Basic Protocol 3: Immunoblot Analysis of Peptide‐Mediated Protein Knockdown
  • Alternate Protocol 1: Co‐Treatment of Cells with Sniper Peptide and Noncovalent CPP
  • Support Protocol 1: Assessing Peptide Toxicity with Lactate Dehydrogenase (LDH) Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Synthesis of CPP‐Linked Protein‐Knockdown Peptides

  • Fmoc‐protected amino acids (GL Biochem)
  • DMF (anhydrous, amide free N,N‐dimethylformamide; Protein Technologies)
  • Fmoc‐glutamic acid‐resin (GL Biochem)
  • NMM (4‐Methylmorpholine; Alfa Aesar)
  • HBTU [2‐(1H‐Benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate; GL Biochem]
  • HoBt‐anhydrous (N‐Hydroxybenzotriazole; GL Biochem)
  • 20% (v/v) piperidine in DMF (VWR Canada)
  • TFA cocktail (trifluoroacetic acid; see recipe)
  • DCM (dichloromethane; EMD)
  • tert‐Butyl methyl ether, 99% (Alfa Aesar)
  • Acetonitrile, HPLC Grade (VWR Canada)
  • Prelude peptide synthesizer (Protein Technologies)
  • Centrifuge
  • Chromatography columns
  • Lyophilizer

Basic Protocol 2: Confirmation of Binding Between Peptide and Target Protein

  • Human embryonic kidney 293 (HEK 293) cells (ATCC CRL‐1573)
  • Cell culture medium (see recipe)
  • FLAG‐tagged target protein plasmid
  • HA‐tagged control peptide plasmid (containing PBD but not CTM)
  • Lipofectamine 2000 (Invitrogen, cat. no. 15338‐500)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Lysis buffer (see recipe)
  • Ice
  • Bio‐Rad DC protein assay reagent (Bio‐Rad, cat. no. 500‐0111)
  • Protein G Sepharose beads (GE Healthcare, cat. no. 17‐0618‐01)
  • Protein A Sepharose CL‐4B (GE Healthcare, cat. no. 17‐0780‐01)
  • Normal mouse IgG (Santa Cruz, cat. no sc‐2025)
  • Anti‐HA antibody (Cell Signaling, cat. no 2362)
  • Wash buffer (see recipe)
  • Sample buffer 4× concentrate (see recipe)
  • 10‐cm tissue culture plates
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Rotator
  • 30‐G needles
  • Heat block

Basic Protocol 3: Immunoblot Analysis of Peptide‐Mediated Protein Knockdown

  • Primary cultured rat neurons (cultured in‐house from Sprague Dawley rats)
  • Cell culture medium (see recipe)
  • Tat‐βsyn36CTM (GL Biochem)
  • Negative control peptides Tat‐βsyn36 (nondegradative), Tat‐scrβsyn36‐CTM (nonbinding) (GL Biochem)
  • Ammonium chloride (Sigma, cat. no. A0171)
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • Lysis buffer (see recipe)
  • Ice
  • Bio‐Rad DC Protein Assay Reagent (Bio‐Rad, cat. no. 500‐0111)
  • Sample buffer 4× concentrate (see recipe)
  • 15% SDS‐PAGE gel
  • PageRuler Prestained Protein Ladder (ThermoScientific, cat. no. SM0671)
  • Blocking buffer: 5% (w/v) skim milk in TBST (see recipe for TBST)
  • TBST (see recipe)
  • Anti‐α‐synuclein antibody (BD Transduction Laboratories, cat. no. 610786)
  • Anti‐mouse IgG horseradish peroxidase (Perkin‐Elmer, cat. no. NEF8822001EA)
  • Anti‐rabbit IgG horseradish peroxidase (Perkin‐Elmer, cat. no. NEF812001EA)
  • Luminata Crescendo ECL (Fisher, cat. no. WBLUR0500)
  • Anti‐β‐actin antibody (Abcam, cat. no. ab8227)
  • 6‐well tissue culture plates
  • 37°C, 5% CO 2 incubator
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Heat block
  • Electrophoresis equipment (see Ursitti et al., )
  • PVDF membranes
  • Immobilon‐P PVDF Transfer Membrane (Millipore, cat. no. IPVH00010)

Alternate Protocol 1: Co‐Treatment of Cells with Sniper Peptide and Noncovalent CPP

  Additional Materials (also see protocol 3)
  • Pep‐1 (Chariot) (Active Motif, cat. no. 30025)
  • Sterile deionized, distilled water
  • Serum‐free medium

Support Protocol 1: Assessing Peptide Toxicity with Lactate Dehydrogenase (LDH) Assay

  Additional Materials (also see protocol 3)
  • In Vitro Toxicology Assay Kit (Sigma, cat. no. TOX7) containing:
    • LDH assay lysis solution
    • LDH assay cofactor solution
    • LDH assay substrate solution
  • Cells incubated in 1 ml cell culture medium (see protocol 3, step 3)
  • Costar 96‐well EIA/RIA plate (Fisher, cat. no. 0720035)
  • Aluminum foil
  • Heated shaker
  • Microplate reader
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Agarraberes, F.A. and Dice, J.F. 2001. A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J. Cell Sci. 114:2491‐2499.
   Amblard, M. , Fehrentz, J.‐A. , Martinez, J. , and Subra, G. 2005. Fundamentals of modern peptide synthesis. Methods Mol. Biol. 298:3‐24.
   Backer, J. , Bourret, L. , and Dice, J.F. 1983. Regulation of catabolism of microinjected ribonuclease A requires the amino‐terminal 20 amino acids. Proc. Natl. Acad. Sci. U.S.A. 80:2166‐2170.
   Banaszynski, L.A. , Chen, L.‐C. , Maynard‐Smith, L.A. , Ooi, A.G.L. , and Wandless, T.J. 2006. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126:995‐1004.
   Banaszynski, L.A. , Sellmyer, M.A. , Contag, C.H. , Wandless, T.J. , and Thorne, S.H. 2008. Chemical control of protein stability and function in living mice. Nat. Med. 14:1123‐1127.
   Bedell, V.M. , Wang, Y. , Campbell, J.M. , Poshusta, T.L. , Starker, C.G. , Krug, R.G. II , Tan, W. , Penheiter, S.G. , Ma, A.C. , Leung, A.Y.H. , Fahrenkrug, S.C. , Carlson, D.F. , Voytas, D.F. , Clark, K.J. , Essner, J.J. , and Ekker, S.C. 2013. In vivo genome editing using a high‐efficiency TALEN system. Nature 490:114‐118.
   Bonger, K.M. , Chen, L.‐C. , Liu, C.W. , and Wandless, T.J. 2011. Small‐molecule displacement of a cryptic degron causes conditional protein degradation. Nat. Chem. Biol. 7:531‐537.
   Bonger, K.M. , Rakhit, R. , Payumo, A.Y. , Chen, J.K. , and Wandless, T.J. 2014. General method for regulating protein stability with light. ACS Chem. Biol. 9:111‐115.
   Castanotto, D. and Rossi, J.J. 2009. The promises and pitfalls of RNA‐interference‐based therapeutics. Nature 457:426‐433.
   Chen, X. , Vinade, L. , Leapman, R.D. , Petersen, J.D. , Nakagawa, T. , Phillips, T.M. , Sheng, M. , and Reese, T.S. 2005. Mass of the postsynaptic density and enumeration of three key molecules. Proc. Natl. Acad. Sci. U.S.A. 102:11551‐11556.
   Cheng, D. , Hoogenraad, C.C. , Rush, J. , Ramm, E. , Schlager, M.A. , Duong, D.M. , Xu, P. , Wijayawardana, S.R. , Hanfelt, J. , Nakagawa, T. , Sheng, M. , and Peng, J. 2006. Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum. Mol. Cell. Proteom. 5:1158‐1170.
   Chiang, H.L. , Terlecky, S.R. , Plant, C.P. , and Dice, J.F. 1989. A role for a 70‐kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246:382‐385.
   Cong, L. , Ran, F.A. , Cox, D. , Lin, S. , Barretto, R. , Habib, N. , Hsu, P.D. , Wu, X. , Jiang, W. , Marraffini, L.A. , and Zhang, F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339:819‐823.
   Cuervo, A.M. and Dice, J.F. 2000. Unique properties of lamp2a compared to other lamp2 isoforms. J. Cell Sci. 113:4441‐4450.
   Cuervo, A.M. and Wong, E. 2013. Chaperone‐mediated autophagy: roles in disease and aging. Cell 24:92‐104.
   Dice, J.F. 1990. Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem. Sci. 15:305‐309.
   Fan, X. , Jin, W.Y. , Lu, J. , Wang, J. , and Wang, Y.T. 2014. Rapid and reversible knockdown of endogenous proteins by peptide‐directed lysosomal degradation. Nat. Neurosci. 17:471‐480.
   Gaj, T. , Gersbach, C.A. , and Barbas, C.F. III. 2013. ZFN, TALEN, and CRISPR/Cas‐based methods for genome engineering. Trends Biotechnol. 31:397‐405.
   Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Protein Sci. 68:10.1.1‐10.1.44.
   Goldberg, A.L. 2003. Protein degradation and protection against misfolded or damaged proteins. Nature 426:895‐899.
   Hill, M.D. , Martin, R.H. , Mikulis, D. , Wong, J.H. , Silver, F.L. , Terbrugge, K.G. , Milot, G. , Clark, W.M. , Macdonald, R.L. , Kelly, M.E. , Boulton, M. , Fleetwood, I. , McDougall, C. , Gunnarsson, T. , Chow, M. , Lum, C. , Dodd, R. , Poublanc, J. , Krings, T , Demchuk, A.M. , Goyal, M. , Anderson, R. , Bishop, J. , Garman, D. , Tymianski, M. ; and ENACT trial investigators. 2012. 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.
   Hyde, C. , Johnson, T. , and Sheppard, R.C. 1992. Internal aggregation during solid phase peptide synthesis. Dimethyl sulfoxide as a powerful dissociating solvent. J. Chem. Soc. Chem. Commun. 1573.
   Kanasty, R. , Dorkin, J.R. , Vegas, A. , and Anderson, D. 2013. Delivery materials for siRNA therapeutics. Nat. Materials 12:967‐977.
   Kaushik, S. and Cuervo, A.M. 2012. Chaperone‐mediated autophagy: A unique way to enter the lysosome world. Trends Cell Biol. 22:407‐417.
   Kim, H. and Kim, J.‐S. 2014. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15:321‐334.
   Koga, H. , Martinez‐Vicente, M. , Macian, F. , Verkhusha, V.V. , and Cuervo, A.M. 2011. A photoconvertible fluorescent reporter to track chaperone‐mediated autophagy. Nature Commun. 2:386.
   Kole, R. , Krainer, A.R. , and Altman, S. 2012. RNA therapeutics: Beyond RNA interference and antisense oligonucleotides. Nat. Rev. Drug Discov. 11:125‐140.
   Mery, J. , Granier, C. , Juin, M. , and Brugidou, J. 1993. Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides. Int. J. Pept. Protein Res. 42:44‐52.
   Morris, M.C. , Chaloin, L. , Mery, J. , Heitz, F. , and Divita, G. 1999. A novel potent strategy for gene delivery using a single peptide vector as a carrier. Nucleic Acids Res. 27:3510‐3517.
   Morris, M.C. , Depollier, J. , Mery, J. , Heitz, F. , and Divita, G. 2001. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19:1173‐1176.
   Morris, M.C. , Deshayes, S. , Heitz, F. , and Divita, G. 2012. Cell‐penetrating peptides: From molecular mechanisms to therapeutics. Biol. Cell 100:201‐217.
   Neklesa, T.K. and Crews, C.M. 2012. Greasy tags for protein removal. Nature 487:308‐309.
   Neklesa, T.K. , Tae, H.S. , Schneekloth, A.R. , Stulberg, M.J. , Corson, T.W. , Sundberg, T.B. , Raina, K. , Holley, S.A. , and Crews, C.M. 2011. Small‐molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7:538‐543.
   Ravid, T. and Hochstrasser, M. 2008. Diversity of degradation signals in the ubiquitin–proteasome system. Nat. Rev. Mol. Cell Biol. 9:679‐689.
   Sakamoto, K.M. 2003. Development of protacs to target cancer‐promoting proteins for ubiquitination and degradation. Mol. Cell. Proteom. 2:1350‐1358.
   Salvador, N. , Aguado, C. , Horst, M. , and Knecht, E. 2000. Import of a cytosolic protein into lysosomes by chaperone‐mediated autophagy depends on its folding state. J. Biol. Chem. 275:27447‐27456.
   Shamloo, M. , Soriano, L. , Wieloch, T. , Nikolich, K. , Urfer, R. , and Oksenberg, D. 2005. Death‐associated protein kinase is activated by dephosphorylation in response to cerebral ischemia. J. Biol. Chem. 280:42290‐42299.
   Slot, L.A. , Lauridsen, A.‐M. , and Hendil, K. 1986. Intracellular protein degradation in serum‐deprived human fibroblasts. J. Biochem. 237:491‐498.
   Sugiyama, Y. , Kawabata, I. , Sobue, K. , and Okabe, S. 2005. Determination of absolute protein numbers in single synapses by a GFP‐based calibration technique. Nat. Methods 2:677‐684.
   Tu, W. , Xu, X. , Peng, L. , Zhong, X. , Zhang, W. , Soundarapandian, M.M. , Belal, C. , Wang, M. , Jia, N. , Zhang, W. , Lew, F. , Chan, S.L. , Chen, Y. , and Lu, Y. 2010. DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke. Cell 140:222‐234.
   Ursitti, J.A. , Mozdzanowski, J. , and Speicher, D.W. 2001. Electroblotting from polyacrylamide gels. Curr. Protoc. Protein Sci. 00:10.7.1‐10.7.14.
   Vivès, E. , Brodin, P. , and Lebleu, B. 1997. 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.
PDF or HTML at Wiley Online Library