Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors or drugs to intact cells. One limitation of CPPs is their rapid degradation by intracellular proteases. β‐hairpin “protectides” have previously been demonstrated to be long‐lived under cytosolic conditions due to their secondary structure. The goal of this work was to demonstrate that arginine‐rich β‐hairpin peptides function as both protectides and as CPPs. Peptides exhibiting a β‐hairpin motif were found to be rapidly internalized into cells with their uptake efficiency dependent on the number of arginine residues in the sequence. Cellular internalization of the β‐hairpin peptides was compared to unstructured, scrambled sequences and to commercially available, arginine‐rich CPPs. The unstructured peptides displayed greater uptake kinetics compared to the structured β‐hairpin sequences; however, intracellular stability studies revealed that the β‐hairpin peptides exhibited superior stability under cytosolic conditions with a 16‐fold increase in peptide half‐life. This study identifies a new class of long‐lived CPPs that can overcome the stability limitations of peptide‐based reporters or bioactive delivery mechanisms in intact cells.
Most cell penetrating peptides (CPPs) are unstructured and susceptible to proteolytic degradation. One alternative is to incorporate D‐chirality amino acids into unstructured CPPs to allow for enhanced uptake and intracellular stability. This work investigates CPP internalization using a series of time, concentration, temperature, and energy dependent studies, resulting in a three‐fold increase in uptake and 50‐fold increase in stability of D‐chirality peptides over L‐chirality counterparts. CPP internalization occurred via a combination of direct penetration and endocytosis, with a percentage of internalized CPP expelling from cells in a time‐dependent manner. Mechanistic studies identified that cells exported the intact internalized D‐chirality CPPs via an exocytosis independent pathway, analogous to a direct penetration method out of the cells. These findings highlight the potential of a D‐chirality CPP as bio‐vector in therapeutic and biosensing applications, but also identify a new expulsion method suggesting a relationship between uptake kinetics, intracellular stability, and export kinetics.
more » « less- Award ID(s):
- 1846900
- NSF-PAR ID:
- 10455560
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- AIChE Journal
- Volume:
- 67
- Issue:
- 1
- ISSN:
- 0001-1541
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)The discovery of cell penetrating peptides (CPPs) with unique membrane activity has inspired the design and synthesis of a variety of cell penetrating macromolecules, which offer tremendous opportunity and promise for intracellular delivery of a variety of imaging probes and therapeutics. While cell penetrating macromolecules can be designed and synthesized to have equivalent or even superior cell penetrating activity compared with natural CPPs, most of them suffer from moderate to severe cytotoxicity. Inspired by recent advances in peptide self-assembly and cell penetrating macromolecules, in this work, we demonstrated a new class of peptide assemblies with intrinsic cell penetrating activity and excellent cytocompatibility. Supramolecular assemblies were formed through the self-assembly of de novo designed multidomain peptides (MDPs) with a general sequence of K x (QW) 6 E y in which the numbers of lysine and glutamic acid can be varied to control supramolecular assembly, morphology and cell penetrating activity. Both supramolecular spherical particles and nanofibers exhibit much higher cell penetrating activity than monomeric MDPs while supramolecular nanofibers were found to further enhance the cell penetrating activity of MDPs. In vitro cell uptake results suggested that the supramolecular cell penetrating nanofibers undergo macropinocytosis-mediated internalization and they are capable of escaping from the lysosome to reach the cytoplasm, which highlights their great potential as highly effective intracellular therapeutic delivery vehicles and imaging probes.more » « less
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Abstract Background information Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B‐type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools.
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aly1 ∆aly2 ∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α‐arrestin Aly1 by the protein phosphatase calcineurin improves steady‐state and substrate‐induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α‐arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol‐3‐phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3H]‐inositol‐labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a‐arrestins in this regulation.Conclusions The α‐arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance.
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