Novel cell‐penetrating peptide targeting mitochondria

Abstract

Cell-penetrating peptides (CPPs) are short, nontoxic peptides with cationic and/or amphipathic properties able to cross the cellular membrane. CPPs are used for the delivery of a wide variety of cargoes, such as proteins, oligonucleotides, and therapeutic molecules. The aim of the present study was to synthesize unusually small novel CPPs targeting mitochondria based on the Szeto-Schiller peptide (SS-31) to influence intramitochondrial processes and to improve the biologic effects. All the peptides used were synthesized manually using 9-fluorenylmethyloxycarbonyl chemistry. In the first part of the study, HeLa 705, U87, and bEnd.3 cells were used as in vitro delivery model. Cells were incubated for 24 h at 37°C and 5% CO2 with different concentrations of our peptides. Cell proliferation assay was performed to evaluate cell viability. Biologic effects such as mitochondrial membrane potential and antioxidant activity were evaluated. H2O2 was used as positive control. Uptake studies were performed using peptides conjugated with 5(6)-carboxyfluorescein (FAM). Fluorescent microscopy was used to determine presence and localization of peptides into the cells. Isolated mitochondria from pretreated cells and mitochondria treated after isolation were used to confirm the targeting ability of the peptide. Uptake of FAM alone was used as negative control. Microscopy studies confirmed the ability of peptides to penetrate cell. Localization analysis showed increase in uptake by 35% compared with SS-31. Mitochondrial CPP 1 (mtCPP-1) had no effect on mitochondrial membrane potential and prevented reactive oxygen species formation in bEnd.3 cells by 2-fold compared with SS-31. No cytotoxicity was observed even at high concentration (100 µM). These data suggest that mtCPP-1 is a mitochondrial CPP and protect mitochondria from oxidative damage due to its own antioxidant activities.

Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem

“…The predominant approach for targeting CPP-bound cargo calls for the fusion of canonical signal sequences to cargo proteins, or even to the CPP tag itself (reviewed in (54)). Using this methodology, cargos have been delivered to the nucleus (55, 56), nucleolus (57), lysosome (58), peroxisome (55), the mitochondria (55, 59, 60) and the endoplasmic reticulum (55, 61), to name a few. Drawbacks to this approach include decreased uptake of tagged CPPs (61) and potential endosomal entrapment en route to the intended compartment.…”

Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial Cell-Penetrating Peptide and a Glutathione Analog Peptide

“…We have previously shown that mtCPP1 and UPF25 did not affect the viability of different cell lines even at high concentration (100 μM) 27, 28. We compared the effects of mtgCPP at different concentrations on the viability of HeLa 705 cells after 24 hr (Figure 2).…”

“…All peptides were designed based on our previously published peptides, mtCPP1 and UPF25 27, 28. Peptides were synthesized in stepwise manner in a 0.1 mmol scale on a Biotage Alstra Plus peptide synthesizer (Biotage) using a fluorenylmethyloxycarbonyl (fmoc) solid-phase peptide synthesis (SPPS) strategy with ChemMatrix Rink Amide resin (0.45 mmol/g) as a solid support to obtain C-terminally amidated peptides 30 .…”

“…The inspiration came from the SS31 peptide, which is a tetrapeptide with alternating aromatic residues and basic amino acids, and we could demonstrate a 2-fold better superoxide anion scavenging ability for mtCPP1 compared with SS31 27 . We have also designed the GSH analog peptide 25 (UPF25) by adding the non-coded amino acid O-methyl-L-tyrosine (Tyr(Me)) to the N terminus, changing the γ-glutamate to α-glutamate, and amidation of the C terminus to GSH (Figure 1B).…”

Perinatal Hypoxic-Ischemic Encephalopathy and Neuroprotective Peptide Therapies: A Case for Cationic Arginine-Rich Peptides (CARPs)

“…In addition to neuronal cell surface receptor interactions, CARPs may also exert pleiotropic neuroprotective effects by targeting mitochondria and maintaining mitochondrial integrity by reducing calcium influx into the organelle [65,77,78,79,80,81,82,83]. CARPs have the capacity to inhibit proteolytic enzymes including the proteasome [84,85], as well as pro-protein convertases [86,87,88] that activate MMPs [89].…”