ABCMdb

PMID: 21625534

Johnson JS, Gentzsch M, Zhang L, Ribeiro CM, Kantor B, Kafri T, Pickles RJ, Samulski RJ
AAV exploits subcellular stress associated with inflammation, endoplasmic reticulum expansion, and misfolded proteins in models of cystic fibrosis.
PLoS Pathog. 2011 May;7(5):e1002053. Epub 2011 May 19., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Gly551Asp ABCC7 p.Asp572Asn By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. Login to comment 125 ABCC7 p.Gly551Asp ABCC7 p.Asp572Asn Western blot of proteins expressed in BHK-21 cell lines depicting level of glycosylation of CFTR, and mutants DF508 (misfolded), G551D (properly folded), D572N (misfolded), 14106(intermediate folding defect). Login to comment 133 ABCC7 p.Gly551Asp The G551D (glycine to aspartic acid) CFTR mutant folds properly and traffics to the plasma membrane, but does not display an operative chloride ion channel [34]. Login to comment 134 ABCC7 p.Gly551Asp When AAV2 was administered to cell lines expressing G551D-CFTR (Fig. 4C), no significant difference in transduction was observed compared to control or CFTR-expressing cells (Fig. 4D). Login to comment 136 ABCC7 p.Asp572Asn D572N (aspartic acid to asparagine) CFTR is misfolded, rapidly cleared from the cell, and cannot be rescued even at low temperature, whereas the truncated 1410X-CFTR mutant shows a partial defect in trafficking [35]. Login to comment 137 ABCC7 p.Asp572Asn Based on measured levels of transduction, AAV is able to exploit expression of D572N-CFTR to levels similar to that achieved in DF508-CFTR-expressing cells. Login to comment 141 ABCC7 p.Gly551Asp Together, these data suggest that AAV is able to exploit cells expressing a variety of misfolded proteins. Moreover, this exploitation is not related to loss of wild-type CFTR activity at the plasma membrane, since G551D-CFTR-expressing cells did not show increased AAV transduction even in the absence of ion channel function. Login to comment 227 ABCC7 p.Gly551Asp Additionally, using a panel of mutant CFTR proteins we show this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity, since the G551D-CFTR mutant did not confer increased susceptibility to AAV infection. Login to comment 283 ABCC7 p.Gly551Asp ABCC1 p.Asp572Asn Materials and Methods Cell culture HeLa cells were maintained at 37uC and 5% CO2 in Dulbecco`s modified Eagle`s medium (DMEM) that was supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin. BHK-21 cells were grown at 37uC and 5% CO2 in Dulbecco`s modified Eagle`s medium (DMEM) and Ham`s F12 medium (50:50) that was supplemented with 5% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin. BHK-21 clones expressing wild-type CFTR, CFTR-DF508, CFTR-G551D, CFTR-D572N, CFTR- 1410X, wild-type MRP1, or MRP1-DF728 were maintained in selection medium containing methotrexate (250 mg/ml). Login to comment