Document Detail


Cell penetration peptides for enhanced entry of αB-crystallin into lens cells.
MedLine Citation:
PMID:  23150610     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
PURPOSE: The prevalence of cataract increases with age. Conversely, the abundance of native α-crystallin diminishes with age and cataract development. We hypothesize replenishing lens α-crystallin may delay or prevent cataract. Herein we investigated the ability of cell penetration peptides (CPP) to enhance entry of α-crystallins into lens-derived cells.
METHODS: Recombinant αB-crystallins were modified by the addition of CPPs. Candidate CPP were designed with reference to the HSV-1 glycoprotein C gene (gC) or the HIV-1 TAT peptide. αB-crystallins produced by fusing gC or TAT were over-expressed in E. coli. Purified proteins were subjected to size exclusion chromatography (SEC) to characterize oligomeric complexes (OC). Chaperone-like activity (CLA) was evaluated by measuring the ability of α-crystallins to suppress chemically-induced protein aggregation. To evaluate protein uptake, labeled α-crystallins were incubated with HLE B3 cells and monitored by fluorescence microscopy for 48 hours.
RESULTS: We examined the effects of the addition of CPP on the structure, CLA, and cell transduction properties of αB-crystallins. C-terminal CPP fused crystallins had poor solubility. In contrast, N-terminal tagged αB-crystallins were soluble. These modified αB-crystallins formed OC that were larger than wild-type based on SEC. Wild-type and gC tagged αB-crystallin displayed robust CLA. Subunit exchange was observed when gC-fused αB-crystallin was mixed with αA. In contrast to wild-type, modified α-crystallins accumulated in HLE B3 cells.
CONCLUSIONS: Addition of CPP improves the uptake of αB-crystallins into HLE B3 cells. No undesirable changes to the chaperone-like abilities of α-crystallins were observed in αB-crystallin modified by the addition of the gC-derived CPP.
Authors:
Niklaus H Mueller; David A Ammar; J Mark Petrash
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-01-02
Journal Detail:
Title:  Investigative ophthalmology & visual science     Volume:  54     ISSN:  1552-5783     ISO Abbreviation:  Invest. Ophthalmol. Vis. Sci.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-03     Completed Date:  2013-02-21     Revised Date:  2013-07-11    
Medline Journal Info:
Nlm Unique ID:  7703701     Medline TA:  Invest Ophthalmol Vis Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2-8     Citation Subset:  IM    
Affiliation:
Department of Ophthalmology, University of Colorado, School of Medicine, Aurora, CO, USA.
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MeSH Terms
Descriptor/Qualifier:
Biological Transport
Cell-Penetrating Peptides / genetics,  metabolism*
Cells, Cultured
Chromatography, Gel
Electrophoresis, Polyacrylamide Gel
Epithelial Cells / metabolism*
Escherichia coli / genetics
Fluorescent Antibody Technique, Indirect
Gene Expression Regulation / physiology
Genetic Vectors
Humans
Lens, Crystalline / metabolism*
Microscopy, Confocal
Molecular Chaperones / metabolism
Recombinant Fusion Proteins / genetics,  metabolism*
Transduction, Genetic
Viral Envelope Proteins / genetics
alpha-Crystallin B Chain / genetics,  metabolism*
tat Gene Products, Human Immunodeficiency Virus / genetics
Grant Support
ID/Acronym/Agency:
5RC1EY020361/EY/NEI NIH HHS; P30 DK57516/DK/NIDDK NIH HHS; UL1RR025780/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
0/Cell-Penetrating Peptides; 0/Molecular Chaperones; 0/Recombinant Fusion Proteins; 0/Viral Envelope Proteins; 0/alpha-Crystallin B Chain; 0/glycoprotein gC, herpes simplex virus type 1; 0/tat Gene Products, Human Immunodeficiency Virus
Comments/Corrections

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