αB-Crystallin Peptide Fused with Elastin-like Polypeptide: Intracellular Activity in Retinal Pigment Epithelial Cells Challenged with Oxidative Stress
Sara Aly Attia,
Anh Tan Truong,
Alvin Phan,
Shin-Jae Lee,
Manal Abanmai,
Marinella Markanovic,
Hugo Avila,
Haozhong Luo,
Atham Ali,
Parameswaran G. Sreekumar,
Ram Kannan,
J. Andrew MacKay
Affiliations
Sara Aly Attia
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Anh Tan Truong
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Alvin Phan
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Shin-Jae Lee
Mann Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, USA
Manal Abanmai
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Marinella Markanovic
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Hugo Avila
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Haozhong Luo
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Atham Ali
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Parameswaran G. Sreekumar
Doheny Eye Institute, Pasadena, CA 91103, USA
Ram Kannan
Doheny Eye Institute, Pasadena, CA 91103, USA
J. Andrew MacKay
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
Background: Oxidative stress-induced retinal degeneration is among the main contributing factors of serious ocular pathologies that can lead to irreversible blindness. αB-crystallin (cry) is an abundant component of the visual pathway in the vitreous humor, which modulates protein and cellular homeostasis. Within this protein exists a 20 amino acid fragment (mini-cry) with both chaperone and antiapoptotic activity. This study fuses this mini-cry peptide to two temperature-sensitive elastin-like polypeptides (ELP) with the goal of prolonging its activity in the retina. Methods: The biophysical properties and chaperone activity of cry-ELPs were confirmed by mass spectrometry, cloud-point determination, and dynamic light scattering ’DLS’. For the first time, this work compares a simpler ELP architecture, cry-V96, with a previously reported ELP diblock copolymer, cry-SI. Their relative mechanisms of cellular uptake and antiapoptotic potential were tested using retinal pigment epithelial cells (ARPE-19). Oxidative stress was induced with H2O2 and comparative internalization of both cry-ELPs was made using 2D and 3D culture models. We also explored the role of lysosomal membrane permeabilization by confocal microscopy. Results: The results indicated successful ELP fusion, cellular association with both 2D and 3D cultures, which were enhanced by oxidative stress. Both constructs suppressed apoptotic signaling (cleaved caspase-3); however, cry-V96 exhibited greater lysosomal escape. Conclusions: ELP architecture is a critical factor to optimize delivery of therapeutic peptides, such as the anti-apoptotic mini-cry peptide; furthermore, the protection of mini-cry via ELPs is enhanced by lysosomal membrane permeabilization.
