Enhancing the Antimicrobial Properties of Peptides through Cell-Penetrating Peptide Conjugation: A Comprehensive Assessment.

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus, at concentrations ranging from 3 to 12 µM, exhibited enhanced antimicrobial activity against B. cereus. TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa, at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli (E. coli) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus, MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development.

Genomic DNA recombination with cell-penetrating peptide-tagged cre protein in mouse skeletal and cardiac muscle.

The Cre-loxP recombination system has been used to promote DNA recombination both in vitro and in vivo. For in vivo delivery, Cre expression is commonly achieved through the use of tissue/cell type-specific promoters, viral infection, or drug inducible transcription and protein translocation to promote targeted DNA excision. The development of cell permeable (or penetrating) peptide tagged proteins has facilitated the delivery of Cre recombinase protein into cells in culture, organotypic slide culture, or in living animals. In this report, we generated bacterially expressed, his-tagged Cre protein with either a cardiac targeting peptide or an antennapedia peptide at the C-terminus and demonstrated efficient uptake and recombination in both cell culture and mice. To facilitate delivery to cardiac and skeletal muscle, we mixed proteins with pluronic F-127 hydrogel and delivered Cre protein into reporter Rosa26mTmG mouse skeletal muscle or Rosa26LacZ cardiac muscle via ultrasound guided injection. Activation of reporter gene expression indicated that these Cre proteins were enzymatically active. Recombination events were detected only in the vicinity of injection areas. In conclusion, we have developed a method to deliver enzymatically active Cre protein locally to skeletal muscle and cardiac muscle that may be adapted for use with other proteins.

Caveolin-1 scaffolding domain residue phenylalanine 92 modulates Akt signaling.

Caveolin-1 (Cav-1), the homo-oligomeric coat protein of cholesterol-rich caveolae signalosomes, regulates signaling proteins including endothelial nitric oxide synthase (eNOS). The Cav-1 scaffolding domain (a.a. 82-101) inhibits activated eNOS from producing vascular protective nitric oxide (NO), an enzymatic process involving trafficking and phosphorylation. However, we demonstrated that Cav-1 proteins and peptides bearing F92A substitution (CAV(F92A)) could promote cardioprotective NO, most likely by preventing inhibition of eNOS by Cav-1. Herein, we showed that wild-type CAV sequence could, similar to CAV(F92A), stimulate basal NO release, indicating a need to better characterize the importance of F92 in the regulation of eNOS by Cav-1/CAV. To reduce uptake sequence-associated effects, we conjugated a wild-type CAV derivative (CAV(WT)) or a F92A variant (CAV(F92A)) to antennapedia peptide (AP) or lipophilic myristic acid (Myr) and compared their effect on eNOS regulation in endothelial cells. We observed that both CAV(WT) and CAV(F92A) could increase basal NO release, although F92A substitution potentiates this response. We show that F92A substitution does not influence peptide uptake, endogenous Cav-1 oligomerization status and Cav-1 and eNOS distribution to cholesterol-enriched subcellular fractions. Instead, F92A substitution in CAV(WT) influences Akt activation and downstream eNOS phosphorylation status. Furthermore, we show that the cell permeabilization sequence could alter subcellular localization of endogenous proteins, an unexpected way to target different protein signaling cascades. Taken together, this suggests that we have identified the basis for two different pharmacophores to promote NO release; furthermore, there is a need to better characterize the effect of uptake sequences on the cellular trafficking of pharmacophores.