Peptide-mediated Protein Knockdown Technology

protein knockdown technology lysosome no titleTechniques that regulate the level of protein expression by targeting genes at the DNA or RNA level have proven to be powerful strategies in the drive to understand protein expression and function. However, because of their very nature, these techniques are restricted in terms of speed, specificity and reversibility. For instance, these genetic methods of disrupting protein expression can take days to weeks; consequently, cellular and molecular compensation may occur, thereby obscuring expected phenotypes. In addition, as these genetic manipulations result in the eradication of all mRNA splice isoforms, as well as post-translationally modified versions of targeted proteins, these methods lack specificity and are largely limited to studying context-dependent protein function. Finally, the reversibility of these genetic manipulations, including many recently developed on-and-off inducible methods, is relatively slow (being achieved on a timescale of days to weeks) and is incomplete. These limitations, in turn, severely constrain the potential of these methods as both research tools and clinical therapeutics.

Here we present a simple, non-virally mediated, cell membrane–permeant, targeting peptide–based system to rapidly and reversibly knock down an endogenous protein of interest by targeting it for lysosomal degradation (Fan et al., Nature Neuroscience, 2014). As shown below, the peptide consists of a cell membrane-penetrating sequence (roughly 10aa long) that can deliver the peptide across the blood-brain barrier and the plasma membrane of cells; a short target protein-binding amino acid stretch (roughly 10aa long) that specifically bind to the target protein of interest with high affinity; and the chaperone-mediated autophagy-targeting motif (CTM) (5-14 aa long) that can direct the peptide-protein complex for lysosomal degradation.

 degradation peptide

Using this technology, we have designed multiple membrane-permeant peptides that can acutely and reversibly knock down many proteins of interest. Here are the examples of two of our knockdown peptides that target death-associated protein kinase 1 (DAPK1), a vital protein mediating cell death cascades in ischemic stroke, and α-synuclein, a widely-studied protein important for the pathogenesis of Parkinson’s disease, respectively. Based on our great expertise in peptide drug development, we are also open to any kind of collaboration in developing small membrane-permeant peptide drugs that can knock down any pathological protein of interest.

peptide knockdown technology examples