ABCMdb

PMID: 24416359

Cebotaru L, Rapino D, Cebotaru V, Guggino WB
Correcting the cystic fibrosis disease mutant, A455E CFTR.
PLoS One. 2014 Jan 8;9(1):e85183. doi: 10.1371/journal.pone.0085183. eCollection 2014., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Ala455Glu Correcting the Cystic Fibrosis Disease Mutant, A455E CFTR Liudmila Cebotaru1,2 , Daniele Rapino1,2 , Valeriu Cebotaru3 , William B. Guggino2 * 1 Department of Ophthalmology, School of Medicine, The Johns Hopkins University, Baltimore, Maryland, United States of America, 2 Department of Physiology, School of Medicine, The Johns Hopkins University, Baltimore, Maryland, United States of America, 3 Department of Medicine, School of Medicine, The Johns Hopkins University, Baltimore, Maryland, United States of America Abstract Cystic fibrosis is caused by more than 1000 mutations, the most common being the DF508 mutation. Login to comment 2 ABCC7 p.Ala455Glu Here we have studied the class V mutation A455E. Login to comment 3 ABCC7 p.Ala455Glu We report that the mature and immature bands of A455E are rapidly degraded primarily by proteasomes; the short protein half-life of this mutant therefore resembles that of DF508 CFTR. Login to comment 4 ABCC7 p.Ala455Glu A455E could be rescued by treatment of the cells with proteasome inhibitors. Login to comment 5 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Furthermore, co-transfection of A455E with the truncation mutant D264 CFTR also rescued the mature C band, indicating that A455E can be rescued by transcomplementation. Login to comment 6 ABCC7 p.Ala455Glu We found that D264 CFTR bound to A455E, forming a bimolecular complex. Login to comment 7 ABCC7 p.Ala455Glu Treatment with the compound correctors C3 and C4 also rescued A455E. Login to comment 8 ABCC7 p.Ala455Glu These results are significant because they show that although DF508 belongs to a different class than A455E, it can be rescued by the same strategies, offering therapeutic promise to patients with Class V mutations. Login to comment 9 ABCC7 p.Ala455Glu Citation: Cebotaru L, Rapino D, Cebotaru V, Guggino WB (2014) Correcting the Cystic Fibrosis Disease Mutant, A455E CFTR. Login to comment 23 ABCC7 p.Gly551Asp Class III mutations produce a protein that has defective regulation; the most common is the G551D mutation, which reaches the cell surface but does not conduct chloride [6,7]. Login to comment 26 ABCC7 p.Ala455Glu One of these class V mutations is A455E. Login to comment 27 ABCC7 p.Ala455Glu The A455E mutation is located in NBD1. Login to comment 29 ABCC7 p.Arg117His Unlike other mild missense mutations such as R117H that have altered channel conductance [10] and are considered class IV mutations, the single-channel characteristics of A445E resemble those of wild-type CFTR [11,12] . Login to comment 30 ABCC7 p.Ala455Glu Thus, because the mild disease resulting from A455E is thought to arise from reduced protein expression, it is considered a class V mutation. Login to comment 31 ABCC7 p.Ala455Glu Thus, an effective pharmacological approach to treating this mutation should involve increasing the protein levels of A455E. Login to comment 35 ABCC7 p.Ala455Glu The purpose of the current study was to determine whether analogous transcomplementation can be used to enhance the protein processing of A455E. Login to comment 37 ABCC7 p.Ala455Glu Plasmids and constructs The construct pEGFP A455E was a gift from Dr. Gary Cutting at Johns Hopkins U. Login to comment 49 ABCC7 p.Ala455Glu A455E has reduced expression of mature CFTR. Login to comment 50 ABCC7 p.Ala455Glu Cos7 cells were transfected with 2 mg of wild-type, A455E, or DF508 CFTR constructs. Login to comment 52 ABCC7 p.Ala455Glu Note that there is much less mature C band in the A455E sample than in the wild-type sample. Login to comment 53 ABCC7 p.Ala455Glu In this and subsequent blots, some mature C band was detected with A455E (n = 8). Login to comment 56 ABCC7 p.Ala455Glu Cells transfected with A455E cDNA were treated either with MG132, a more general inhibitor (n = 6) (A, B), or the more specific inhibitor of proteasomes, PS341 (n = 2) (C). Login to comment 57 ABCC7 p.Ala455Glu Note that in both cases, proteasome inhibition caused an increase in both the immature B and mature C bands of A455E. Login to comment 58 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu doi:10.1371/journal.pone.0085183.g002 Results Expression of A455E When we compared the expression of the A455E mutant to that of both wild-type and nF508 CFTR (Fig. 1) by western blotting, we found that the amount of CFTR protein was greatly reduced in the Cos7 cells transfected with the A455E mutant. Login to comment 59 ABCC7 p.Ala455Glu To evaluate the degradation of A455E CFTR, we treated the cells with MG132, a non-specific inhibitor of proteasomal degradation. Login to comment 60 ABCC7 p.Ala455Glu Fig. 2 shows that in the presence of MG132, the protein expression of both the B and C bands of A455E CFTR increased dramatically. Login to comment 62 ABCC7 p.Ala455Glu PS341 also caused an increase in both the B and C bands of A455E. Login to comment 63 ABCC7 p.Ala455Glu Furthermore, we noted that A455E could be rescued by growing the cells at a reduced temperature, as had previously been observed for DF508 [13]. Login to comment 64 ABCC7 p.Ala455Glu In sharp contrast, there was no increase in the protein expression of A455E CFTR when the cells were treated with the aggresome inhibitor tubacin or the lysosomal inhibitor E64 (Fig. 3). Login to comment 65 ABCC7 p.Ala455Glu These data suggest that A455E is degraded primarily in proteasomes. Login to comment 66 ABCC7 p.Ala455Glu To evaluate how rapidly the A455E CFTR protein is degraded, we treated the transfected cells with cycloheximide for between 1 and 7 hours (Fig 4). Login to comment 67 ABCC7 p.Ala455Glu Surprisingly, both the B and C bands of A455E CFTR rapidly disappeared in the cells treated with cycloheximide (Fig. 4), suggesting that it is rapidly degraded, as is DF508 CFTR (see [13]). Login to comment 68 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu n264 CFTR increases the processing of band B to band C of A455E CFTR We then tested whether the truncation mutant, n264 CFTR, was capable of transcomplementation with A455E (Fig. 5). Login to comment 69 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu In order to determine whether D264 CFTR affects the maturation of A455E CFTR, we cotransfected D264 CFTR and A455E CFTR into Cos7 cells and found that the mature C band from A455E CFTR was increased in cells cotransfected with D264 CFTR, as compared to cells transfected with A455E cDNA alone (Fig. 5). Login to comment 72 ABCC7 p.Ala455Glu Cos7 cells were transfected with A455E CFTR cDNA and treated for 16 h with the lysosome inhibitor E64 (n = 4) (A) There was very little change in band density in any of the treated groups or in the presence of the inhibitors when compared to the control or the HDAC6 inhibitor tubacin to inhibit aggresomes (n = 3) (B, C). Login to comment 74 ABCC7 p.Ala455Glu doi:10.1371/journal.pone.0085183.g003 n264 CFTR binds to A455E CFTR, forming a biomolecular complex We and others have shown that transcomplementation can occur via direct binding of truncated forms of CFTR to DF508-CFTR and via chaperone displacement [17]. Login to comment 75 ABCC7 p.Ala455Glu In order to assess these possibilities in A455E CFTR, we conducted co-immunpre- cipitation experiments. Login to comment 76 ABCC7 p.Ala455Glu Fig. 6 shows that D264-CFTR did indeed bind to A455E, in both the absence and presence of the proteasome inhibitor MG123. Login to comment 77 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Correctors 4A (C4) and VX325 (C3) increase the processing of band B to band C of A455E CFTR We next asked whether small-molecule correctors might be effective in rescuing A455E CFTR (Fig. 7). Login to comment 79 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu We found that corrector C4 does have a robust effect on A455E CFTR; in contrast, C3 had only a minimal effect on A455E (Fig. 7). Login to comment 83 ABCC7 p.Ala455Glu Here we show that A455E can also be rescued by transcomplementation. Login to comment 87 ABCC7 p.Ala455Glu The A455E mutation, on the other hand, is located within the F1-type ATP-binding core subdomain near to the ABC protein signature, the Walker A domain [24]. Login to comment 88 ABCC7 p.Ala455Glu Given the proximity to the Walker A domain, one might expect that A455E would have alterations in gating. Login to comment 90 ABCC7 p.Ala455Glu A455E also functions well as a regulator of other channels [25]. Login to comment 93 ABCC7 p.Ala455Glu At least two studies have failed to detect mature band C from A455E [11,26], although the results of their electrophysiological studies suggested that some mature band C must have been present at the plasma membrane in order to generate chloride currents [11]. Login to comment 94 ABCC7 p.Ala455Glu In our study, we do detect some mature band C at the plasma membrane, but when the cells are treated with cycloheximide to evaluate protein degradation, it is clear that the mature band C of A455E is rapidly degraded along with the immature B band. Login to comment 98 ABCC7 p.Ala455Glu have shown that A455E has a pattern of degradation that is clearly different from that of DF508 CFTR. Login to comment 99 ABCC7 p.Ala455Glu A455E appears to be proteolytically cleaved within the NBD1-R domain to form C-terminal aggregates. Login to comment 101 ABCC7 p.Ala455Glu Clearly, these results show that the A455E mutant is distinctly different from DF508 CFTR. Login to comment 102 ABCC7 p.Ala455Glu Nevertheless, A455E shows some similarity to DF508 CFTR. Login to comment 103 ABCC7 p.Ala455Glu Although A455E is uniquely cleaved in the cytoplasm, our results show that it is still degraded in the proteasome, because treatment with two types of proteasome inhibitors led to significant increases in steady-state protein levels. Login to comment 104 ABCC7 p.Ala455Glu In fact, we saw a rather large increase in the mature band of A455E after proteasome inhibition. Login to comment 105 ABCC7 p.Ala455Glu This result is in contrast to the response of A455E to lysosomal inhibitors, which were without effect. Login to comment 107 ABCC7 p.Ala455Glu A455E binds to n27-264 CFTR. Login to comment 108 ABCC7 p.Ala455Glu Cos7 cells were transfected with both D27-264 CFTR and an A455E CFTR construct bearing a GFP tag. Login to comment 109 ABCC7 p.Ala455Glu Anti-GFP antibodies were used to pull down A455E, and the gels were blotted with anti-CFTR antibody. Login to comment 113 ABCC7 p.Ala455Glu Transcomplementation of A455E by D27-264 CFTR. Login to comment 114 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Both the C and B bands of A455E CFTR were increased when cells were cotransfected with D27-264 CFTR, showing that A455E could be rescued by transcomplementation. Login to comment 117 ABCC7 p.Ala455Glu Effect of protein synthesis inhibition on the degradation of A455E. Login to comment 118 ABCC7 p.Ala455Glu Cos7 cells were transfected with A455E cDNA and treated with cycloheximide (25 mg/ml) for the indicated times. Login to comment 119 ABCC7 p.Ala455Glu Note the rapid decay of both the mature C and immature B bands of A455E. Login to comment 122 ABCC7 p.Ala455Glu Taken together, our data suggest that A455E, like DF508 CFTR, is processed by proteasomes. Login to comment 123 ABCC7 p.Ala455Glu D264 CFTR rescues A455E and forms a molecular complex between the two molecules. Login to comment 126 ABCC7 p.Ala455Glu It is possible that a similar interaction occurs between D264 and A455E. Login to comment 131 ABCC7 p.Ala455Glu The observation that A455E is readily rescued by D264 CFTR suggests that transcomplementation may be influencing NBD1 in the region of the Walker sites. Login to comment 132 ABCC7 p.Ala455Glu One then may ask why the interaction with the normal NBD2 of A455E does not rescue its own NBD1. Login to comment 138 ABCC7 p.Ala455Glu Correctors can rescue A455E. Login to comment 139 ABCC7 p.Ala455Glu Cos7 cells were transfected with A455E and treated with the correctors C3 or C4 for 16 h at the specified concentrations. Login to comment 140 ABCC7 p.Ala455Glu Note that C4 had a profound effect on the immature B band of A455E as well as causing an increase in the mature C band (A, B). Login to comment 143 ABCC7 p.Ala455Glu doi:10.1371/journal.pone.0085183.g007 Therapeutic transcomplementation to rescue A455E would require gene transfer via a virus or non-viral particle. Login to comment 150 ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu Our demonstration that both C3 and C4 can rescue A455E suggests that A455E CFTR may be a better candidate for correction with either compound correctors or transcomplementation than is DF508 CFTR. Login to comment