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Delivery of cargo molecules to cell interiors by a cell‐penetrating peptide‐tagged adaptor
Author(s) -
McMurry Jonathan,
Chrestensen Carol,
Nowak Scott,
Salerno John
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.566.12
Subject(s) - endocytosis , endosome , cell penetrating peptide , calmodulin , peptide , cytoplasm , microbiology and biotechnology , chemistry , cell , signal transducing adaptor protein , biophysics , biochemistry , biology , signal transduction , enzyme
The trans‐activating transcriptional activator (TAT) from HIV‐1 was the first cell penetrating peptide described. Other small peptides capable of crossing animal cell membranes have since been discovered or devised (e.g., Antennapedia peptide, PEP‐1). Cargo attached to CPP is also internalized and it is now clear that CPPs engage endocytosis mechanisms, delivering cargo to endosomes from which a fraction escapes to the cytoplasm. We expressed and purified a CPP‐tagged adaptor protein, TAT‐CaM, consisting of calmodulin with a TAT peptide. TAT‐CaM binds ligands as effectively as unmodified calmodulin as assessed by optical biosensing experiments. To assess the ability of TAT‐CaM to mediate protein translocation, neuronal nitric oxide synthase was labeled with DyLite550 and added to cultured BHK cells. After washing, cells were observed with confocal microscopy; in the absence of TAT‐CaM some aggregates of labeled nNOS were observed on the surfaces of the cells but not inside. In the presence of TAT‐CaM, cell exteriors were cleared of labeled nNOS, but the interiors were heavily labeled. Endosomal interiors loaded with labeled nNOS were clearly visible, but significant nNOS escaped into the cytoplasm. CPP tagged adaptors have potential advantages in convenience and safety. Calmodulin is particularly useful in that it can be used in affinity purification of tagged cargo, and releases cargo in the low Ca 2+ cellinterior.