Presentation
Myristic Acid-Trans-Activator of Transcription Dual Conjugation Improves Intracellular Delivery of Protein Kinase C Beta II Peptide Inhibitor Cargo in Isolated Rat Polymorphonuclear Leukocytes
Experimental Biology 2022
(2022)
Abstract
Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). In previous studies, cell-permeable myristic acid conjugated PKCβII inhibitor (myr-PKCβII-) significantly attenuated PMN SO release induced by phorbol 12-myristate 13-acetate (PMA), a diacylglycerol mimetic. Myr-PKCβII- was determined to be superior to unconjugated peptides and nontreated controls, suggesting enhanced intracellular delivery of cargo. We hypothesize that the simple diffusion of myr-conjugation combined with the endocytotic mechanism of trans-activator of transcription (Tat) would optimize the intracellular delivery of PKCβII- cargo compared to myr-conjugation alone. In this study, we tested the concentration-dependent effects of a dual myr-Tat conjugated PKCβII- (myr-Tat-PKCβII-; N-myr-Tat-CC-SLNPEWNET) on intracellular delivery compared to myr-PKCβII-, scrambled myr-Tat-PKCβII- (myr-Tat-PKCβII- scram), unconjugated PKCβII-, and 0.5% dimethyl sulfoxide (DMSO) vehicle control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII- (20μM), myr-Tat-PKCβII- (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr-Tat-PKCβII-scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after PMA stimulation (100nM). The efficacy of intracellular drug delivery was evaluated by the magnitude of PMA-induced PMN SO release attenuation with the PKCβII- cargo. Data were analyzed with ANOVA Fisher's PLSD post-hoc analysis. Myr-Tat-PKCβII- 5μM (n=12, 0.392±0.04), 7.5μM (n=11, 0.397±0.05), 10μM (n=5, 0.211±0.05) and 20μM (n=5, 0.121±0.02) demonstrated a concentration-dependent increase in intracellular delivery compared to DMSO vehicle control (n=84, 0.496±0.02, all p<0.05). Myr-PKCβII- only significantly increased intracellular delivery at the 20μM concentration (n=27, 0.303±0.02, p<0.05) compared to DMSO vehicle control. Intracellular delivery of myr-Tat-PKCβII- 2μM (n=10, 0.436±0.06) and all concentrations of myr-Tat-PKCβII-scram were not significantly different from DMSO vehicle controls. Results suggest that myr-Tat dual conjugation is superior to myr-conjugation alone at intracellular delivery of cell impermeant cargo. Future studies will investigate the concentration-dependent effects of PKCβII- peptide conjugates on PMA-induced PKCβII activity and translocation to membrane targets, such as NOX-2, using immunocytochemistry and western blot analysis.
Disciplines
Publication Date
April, 2022
Location
Philadelphia, PA
DOI
https://doi.org/10.1096/fasebj.2022.36.S1.R6218
Citation Information
Sunit G Singh, Alexis Verwoert, Arjun Nair, Devani Johnson, et al.. "Myristic Acid-Trans-Activator of Transcription Dual Conjugation Improves Intracellular Delivery of Protein Kinase C Beta II Peptide Inhibitor Cargo in Isolated Rat Polymorphonuclear Leukocytes" Experimental Biology 2022 (2022) Available at: http://works.bepress.com/lindon_young/97/