Expressed Cell-penetrating Peptides Can Induce a Bystander Effect, but Passage Through the Secretory Pathway Reduces Protein Transduction Activity

Abstract

Despite advances in vector technology, inefficient gene transfer still limits clinical efficacy of cancer gene therapy. Cell-penetrating peptides (CPPs), such as the basic domain of the transactivator of transcription (Tat) protein of HIV-1, are internalized by intact cells and have been used to deliver purified recombinant proteins. A combination of gene therapy with protein transduction technology could induce a strong bystander effect and represent a platform to deliver proteins to target cells. However, whether expressed CPP can facilitate intercellular trafficking, i.e., a bystander effect, is controversial. Our data suggest that expressed fusion proteins that contain the basic domain of Tat do not induce a detectable bystander effect. However, Tat-fusion proteins that also contain a secretory signal peptide (SP) can induce a bystander effect in vitro, although the in vivo effect is small. Surprisingly, despite the presence of a SP, the bystander effect does not seem to be related to secretion of the fusion protein. In fact, Tat-fusion proteins are secreted very inefficiently, and protein transduction seems largely mediated by fusion proteins that are released by cell lysis. Modification of Tat can improve secretion efficacy and prevent cleavage by the endoprotease furin, but passage through the secretory pathway is associated with reduced transduction activity of Tat-fusion proteins.

Cationic Cell-Penetrating Peptides Are Potent Furin Inhibitors

“…on biological activities other than transport) have been previously cataloged in a recent review [ 2 ] and include a variety of biological effects, such as oxidative stress effects, responsiveness to heparan sulfate, lipid remodeling, and actin rearrangement. Interestingly, the only prior study that has addressed the interaction of CPPs with proprotein convertases concluded that furin may act to inactivate the TAT 7-57 peptide, although furin-mediated inactivation was not directly demonstrated in this work [ 51 ]. Our data support a contradictory conclusion: that TAT4 7-57 acts to inhibit intracellular furin and/or other convertases.…”

Generation of an efficiently secreted, cell penetrating NF‐κB inhibitor

“…Secretion of PTD‐containing molecules has been the goal of several research papers (23–27). However, as these studies unanimously demonstrate, and our initial experiments show, attachment of a PTD domain to a molecule targeted for secretion (such as mouse IgG2a Fc domain) has a dramatic inhibitory effect on their secretion with most (or all) of the protein retained inside the producing cell.…”

“…In this way, the EGFP partner functions as a secretion carrier. In another recent study, the fluorescent mCherry protein was also efficiently secreted when linked to the TAT3 sequence (27). The disadvantage of these strategies is that fluorescent proteins are foreign and therefore immunogenic in vivo (29), and as such are not suitable for clinical use.…”

“…This view was supported by the fact that exogenous MMP1 did not result in a further increase in NF-B inhibition. Interestingly, the results of Shen et al (27) demonstrated that efficiently secreted TAT3-mCherry was inefficient in transducing cells, suggesting that passage through the secretory pathway reduces protein transduction activity. The fact that we observed biological activity with TAT3-NBD indicates that cleavage from the secretion partner, in our case LAP, is particularly important to restore transduction activity.…”

Designed Multifunctional Peptides for Intracellular Targets

“…The TAT peptide sequence GRKKRRQRRRPPQ is, however, easily cleaved by furin. Thus, CPP is not stable enough in vivo for efficient cargo delivery [ 96 ]. Hemmati et al [ 97 ] identified 310 decapeptides with predicted CPP activity in the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).…”