Protein polymer nanoparticles engineered as chaperones protect against apoptosis in human retinal pigment epithelial cells.
αB-Crystallin is a protein chaperone with anti-apoptotic and anti-inflammatory activity that is apically secreted in exosomes by polarized human retinal pigment epithelium. A 20 amino acid mini-peptide derived from residues 73-92 of αB-crystallin protects human retinal pigment epithelial (RPE) cells from oxidative stress, a process involved in the progression of age-related macular degeneration (AMD). Unfortunately, due to its small size, its development as a therapeutic requires a robust controlled release system. To achieve this goal, the αB-crystallin peptide was re-engineered into a protein polymer nanoparticle/macromolecule with the purpose of increasing the hydrodynamic radius/molecular weight and enhancing potency via multivalency or an extended retention time. The peptide was recombinantly fused with two high molecular weight (~40kDa) protein polymers inspired by human tropoelastin. These elastin-like polypeptides (ELPs) include the following: (i) a soluble peptide called S96 and (ii) a diblock copolymer called SI that assembles multivalent nanoparticles at physiological temperature. Fusion proteins, cryS96 and crySI, were found to reduce aggregation of alcohol dehydrogenase and insulin, which demonstrates that ELP fusion did not diminish chaperone activity. Next their interaction with RPE cells was evaluated under oxidative stress. Unexpectedly, H2O2-induced stress dramatically enhanced cellular uptake and nuclear localization of both cryS96 and crySI ELPs. Accompanying uptake, both fusion proteins protected RPE cells from apoptosis, as indicated by reduced caspase 3 activation and TUNEL staining. This study demonstrates the in vitro feasibility of modulating the hydrodynamic radius for small peptide chaperones by seamless fusion with protein polymers; furthermore, they may have therapeutic applications in diseases associated with oxidative stress, such as AMD.
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California Los Angeles, CA; 90033-9121.
Wan
W
Wang
Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, CA 90033.
Parameswaran G
PG
Sreekumar
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California Los Angeles, CA; 90033-9121.
Vinod
V
Valluripalli
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California Los Angeles, CA; 90033-9121.
Pu
P
Shi
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California Los Angeles, CA; 90033-9121.
Jiawei
J
Wang
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, 21218, USA.
Yi-An
YA
Lin
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, 21218, USA.
Honggang
H
Cui
Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, CA 90033.
Ram
R
Kannan
Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033.|Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033.
David R
DR
Hinton
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California Los Angeles, CA; 90033-9121.|Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90033.
J Andrew
JA
MacKay
D021581:Active Transport, Cell Nucleus; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D002626:Chemistry, Pharmaceutical; D019610:Cytoprotection; D003692:Delayed-Action Preparations; D004337:Drug Carriers; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005240:Feasibility Studies; D006801:Humans; D018832:Molecular Chaperones; D050997:Nanomedicine; D053758:Nanoparticles; D018384:Oxidative Stress; D010316:Particle Size; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000473:pathology; D013678:Technology, Pharmaceutical / Q000379:methods*; D013997:Time Factors; D014333:Tropoelastin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D003692:Delayed-Action Preparations; D004337:Drug Carriers; D018832:Molecular Chaperones; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014333:Tropoelastin; D038203:alpha-Crystallin B Chain; C505407:CASP3 protein, human; D053148:Caspase 3
Anti-apoptosis; Assembly; Cell uptake; Chaperone; Elastin-like polypeptide (ELPs)