Enhancing intracellular delivery of protein kinase C beta II via N-Terminus conjugation of myristic acid-trans-activator of transcription to attenuate superoxide release in rat polymorphonuclear leukocytes

Introduction

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). Previous studies have shown that cell-permeable myristic acid-conjugated PKCβII inhibitor (myr-PKCβII-; SLNPEWNET) (20 µM) attenuated phorbol 12-myristate 13-acetate (PMA)-induced PMN SO release when compared to scrambled peptides (myr-Tat-PKCβII- scram; WNPESLNTE), unconjugated peptides and nontreated controls by ~35%. This suggests an enhanced intracellular delivery of PKCβII-. Studies have also shown that SO release is attenuated via the trans-activator of transcription-conjugated (Tat; YGRKKRRQRRR) NOX-2 inhibitor (Nox2ds-Tat) (80 µM) by ~37%. There have not been evaluations of Tat- and myr-Tat-conjugated PKCβII-. We hypothesize that dual myr-Tat-conjugation would enhance the intracellular delivery of PKCβII- and reduce SO release compared to myr-, Tat-, and myr-Tat-PKCβII- scram.

Methods

The study focused on the effects of myr-Tat-PKCβII- on intracellular delivery compared to myr-PKCβII-, Tat -PKCβII-, and myr-Tat-PKCβII- scram, with 0.5% dimethyl sulfoxide (DMSO) vehicle as the control. Sprague-Dawley (SD) male rats (~400g) were anesthetized with isoflurane and given a 0.5% glycogen i.p. injection (16ml), and 16-18hrs later, PMNs were harvested from the peritoneal cavity and incubated for 15min at 37oC with 5μM myr-Tat-PKCβII-, myr-PKCβII-, Tat-PKCβII-, or myr-Tat-PKCβII- scram. PMN SO release was calculated spectrophotometrically by the change in absorbance at 550 nm over 390s via ferricytochrome c reduction after PMA stimulation (100nM). Data were analyzed using ANOVA Fisher’s PLSD post-hoc analysis.

Results

Myr-Tat-PKCβII- (n=20, 0.338±0.024, p 85% in all groups.

Conclusion

The results of the study suggest that intracellular delivery of PKCβII- cargo using myr-Tat dual conjugation is enhanced when compared to myr-, Tat-conjugated PKCβII-, and myr-Tat-PKCβII- scram. Future studies will utilize western blot analysis and immunohistochemistry to assess the translocation and activity of PKCβII- when conjugated with myr-Tat, myr-, or Tat- peptides.

Funding

This study was funded by the National Heart, Lung & Blood Institute (1R43HL160338) SBIR phase I grant that was awarded to Young Therapeutics, LLC. Philadelphia College of Osteopathic Medicine, the Department of Biomedical Sciences, the Division of Research and the Center for Chronic Diseases of Aging, and Young Therapeutics, LLC.