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Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

Deuquet J, Lausch E, Guex N, Abrami L, Salvi S, Lakkaraju A, Ramirez MC, Martignetti JA, Rokicki D, Bonafe L, Superti-Furga A, van der Goot FG - EMBO Mol Med (2011)

Bottom Line: Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level.Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation.Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland.

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C39F and C218R HFS mutations lead to inter-disulphide bond formation and ER retentionA,D. CHO cells were transfected for 48 h with WT or mutants CMG2-V5 constructs. Cell lysates were subjected to immunoprecipitation with an anti-V5 antibody. Samples were analysed by SDS–PAGE—under reducing or non-reducing conditions—and Western blotting with an anti-V5 antibody.B. CHO cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH enzyme and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.C. HeLa cells were transfected with WT or mutant CMG2-V5 cDNA for 24 h prior to fixation and permeabilization, and labelling with anti-V5 monoclonal and anti-calnexin polyclonal antibodies. Bar: 10 µm.E. CHO cells were transiently transfected with WT or mutant CMG2-V5 cDNA. Cells were lysed in immunoprecipitation buffer and 1 µg/ml of PA was added in the buffer for 1 h at 4°C. After immunoprecipitation using an antibody against the V5 tag, samples were analysed by SDS–PAGE followed by Western blotting against PA or the V5 tag.F. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNA. Surface biotinylation was performed as in 4B. Error bars represent the standard deviation (n = 3). Asterisks represent significant difference with respect to WT (p < 0.05).
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fig06: C39F and C218R HFS mutations lead to inter-disulphide bond formation and ER retentionA,D. CHO cells were transfected for 48 h with WT or mutants CMG2-V5 constructs. Cell lysates were subjected to immunoprecipitation with an anti-V5 antibody. Samples were analysed by SDS–PAGE—under reducing or non-reducing conditions—and Western blotting with an anti-V5 antibody.B. CHO cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH enzyme and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.C. HeLa cells were transfected with WT or mutant CMG2-V5 cDNA for 24 h prior to fixation and permeabilization, and labelling with anti-V5 monoclonal and anti-calnexin polyclonal antibodies. Bar: 10 µm.E. CHO cells were transiently transfected with WT or mutant CMG2-V5 cDNA. Cells were lysed in immunoprecipitation buffer and 1 µg/ml of PA was added in the buffer for 1 h at 4°C. After immunoprecipitation using an antibody against the V5 tag, samples were analysed by SDS–PAGE followed by Western blotting against PA or the V5 tag.F. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNA. Surface biotinylation was performed as in 4B. Error bars represent the standard deviation (n = 3). Asterisks represent significant difference with respect to WT (p < 0.05).

Mentions: Upon expression of C39F or C218R CMG2, both the mature and the ER precursor forms were observed under reducing conditions (Fig 6A), but the relative abundance of the precursor was higher than for the WT protein, as even more apparent after EndoH treatment (Fig 6B). Partial ER retention was confirmed by immunofluorescence analysis, where we could detect both plasma membrane and ER staining, as particularly indicated by the staining of the nuclear membrane (illustrated for C39F in Fig 6C).


Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

Deuquet J, Lausch E, Guex N, Abrami L, Salvi S, Lakkaraju A, Ramirez MC, Martignetti JA, Rokicki D, Bonafe L, Superti-Furga A, van der Goot FG - EMBO Mol Med (2011)

C39F and C218R HFS mutations lead to inter-disulphide bond formation and ER retentionA,D. CHO cells were transfected for 48 h with WT or mutants CMG2-V5 constructs. Cell lysates were subjected to immunoprecipitation with an anti-V5 antibody. Samples were analysed by SDS–PAGE—under reducing or non-reducing conditions—and Western blotting with an anti-V5 antibody.B. CHO cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH enzyme and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.C. HeLa cells were transfected with WT or mutant CMG2-V5 cDNA for 24 h prior to fixation and permeabilization, and labelling with anti-V5 monoclonal and anti-calnexin polyclonal antibodies. Bar: 10 µm.E. CHO cells were transiently transfected with WT or mutant CMG2-V5 cDNA. Cells were lysed in immunoprecipitation buffer and 1 µg/ml of PA was added in the buffer for 1 h at 4°C. After immunoprecipitation using an antibody against the V5 tag, samples were analysed by SDS–PAGE followed by Western blotting against PA or the V5 tag.F. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNA. Surface biotinylation was performed as in 4B. Error bars represent the standard deviation (n = 3). Asterisks represent significant difference with respect to WT (p < 0.05).
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Related In: Results  -  Collection

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fig06: C39F and C218R HFS mutations lead to inter-disulphide bond formation and ER retentionA,D. CHO cells were transfected for 48 h with WT or mutants CMG2-V5 constructs. Cell lysates were subjected to immunoprecipitation with an anti-V5 antibody. Samples were analysed by SDS–PAGE—under reducing or non-reducing conditions—and Western blotting with an anti-V5 antibody.B. CHO cell lysates were subjected to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH enzyme and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.C. HeLa cells were transfected with WT or mutant CMG2-V5 cDNA for 24 h prior to fixation and permeabilization, and labelling with anti-V5 monoclonal and anti-calnexin polyclonal antibodies. Bar: 10 µm.E. CHO cells were transiently transfected with WT or mutant CMG2-V5 cDNA. Cells were lysed in immunoprecipitation buffer and 1 µg/ml of PA was added in the buffer for 1 h at 4°C. After immunoprecipitation using an antibody against the V5 tag, samples were analysed by SDS–PAGE followed by Western blotting against PA or the V5 tag.F. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNA. Surface biotinylation was performed as in 4B. Error bars represent the standard deviation (n = 3). Asterisks represent significant difference with respect to WT (p < 0.05).
Mentions: Upon expression of C39F or C218R CMG2, both the mature and the ER precursor forms were observed under reducing conditions (Fig 6A), but the relative abundance of the precursor was higher than for the WT protein, as even more apparent after EndoH treatment (Fig 6B). Partial ER retention was confirmed by immunofluorescence analysis, where we could detect both plasma membrane and ER staining, as particularly indicated by the staining of the nuclear membrane (illustrated for C39F in Fig 6C).

Bottom Line: Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level.Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation.Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne, Global Health Institute, Lausanne, Switzerland.

Show MeSH
Related in: MedlinePlus