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
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Abstract

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

     
 
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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
     
 
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Materials

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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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
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Figures

Videos

Literature Cited

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