Details

Autor(en) / Beteiligte

• Singh, Sunit G.
• Verwoert, Alexis
• Nair, Arjun
• Johnson, Devani
• Humayun, Annam
• Clair, Logan
• DiLisi, Taylor
• Harrell, Kayla
• Dean, Tameka
• Chen, Qian
• Barsotti, Robert
• Young, Lindon

Titel

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

Ist Teil von

• The FASEB journal, 2022-05, Vol.36 (S1), p.n/a

Ort / Verlag

United States: The Federation of American Societies for Experimental Biology

Erscheinungsjahr

2022

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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.