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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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Mutations of Ig-like domain cysteines lead to ER retention of CMG2A,C. HeLa or CHO cells were transfected for 48 h with WT or mutant CMG2-V5 constructs. Cell extracts were analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.B. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNAs. Confluent cells were pulsed with [35S]-methionine and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Biotinylated CMG2 proteins and expression levels were quantified using the Typhoon scanner. For each experiment, biotinylated CMG2 values were normalized to the synthesis levels and expressed as a percentage of WT. Error bars represent the standard deviation (n = 3).D. CHO cell lysates were submitted to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.E. 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 as well as anti-calnexin polyclonal antibodies. The inserts illustrate an enlargement of a specific region. Bar: 10 µm.
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fig04: Mutations of Ig-like domain cysteines lead to ER retention of CMG2A,C. HeLa or CHO cells were transfected for 48 h with WT or mutant CMG2-V5 constructs. Cell extracts were analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.B. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNAs. Confluent cells were pulsed with [35S]-methionine and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Biotinylated CMG2 proteins and expression levels were quantified using the Typhoon scanner. For each experiment, biotinylated CMG2 values were normalized to the synthesis levels and expressed as a percentage of WT. Error bars represent the standard deviation (n = 3).D. CHO cell lysates were submitted to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.E. 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 as well as anti-calnexin polyclonal antibodies. The inserts illustrate an enlargement of a specific region. Bar: 10 µm.

Mentions: Single mutants of the vWA domain cysteines did not drastically affect the migration pattern, i.e. both the precursor and the mature form were observed (Fig 4A), indicating that a significant proportion of the protein was able to exit the ER. Immunofluorescence analysis and surface biotinylation experiments further showed that mutation of Cys-39 or Cys-218 to alanine does not significantly affect targeting of CMG2 to the cell surface (Fig 4B and E).


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)

Mutations of Ig-like domain cysteines lead to ER retention of CMG2A,C. HeLa or CHO cells were transfected for 48 h with WT or mutant CMG2-V5 constructs. Cell extracts were analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.B. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNAs. Confluent cells were pulsed with [35S]-methionine and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Biotinylated CMG2 proteins and expression levels were quantified using the Typhoon scanner. For each experiment, biotinylated CMG2 values were normalized to the synthesis levels and expressed as a percentage of WT. Error bars represent the standard deviation (n = 3).D. CHO cell lysates were submitted to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.E. 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 as well as anti-calnexin polyclonal antibodies. The inserts illustrate an enlargement of a specific region. Bar: 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3377065&req=5

fig04: Mutations of Ig-like domain cysteines lead to ER retention of CMG2A,C. HeLa or CHO cells were transfected for 48 h with WT or mutant CMG2-V5 constructs. Cell extracts were analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.B. HeLa cells were transfected for 48 h with WT or mutant CMG2-V5 cDNAs. Confluent cells were pulsed with [35S]-methionine and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Biotinylated CMG2 proteins and expression levels were quantified using the Typhoon scanner. For each experiment, biotinylated CMG2 values were normalized to the synthesis levels and expressed as a percentage of WT. Error bars represent the standard deviation (n = 3).D. CHO cell lysates were submitted to immunoprecipitation with an anti-V5 antibody, treated with or without EndoH and analysed by SDS–PAGE and Western blotting with an anti-V5 antibody.E. 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 as well as anti-calnexin polyclonal antibodies. The inserts illustrate an enlargement of a specific region. Bar: 10 µm.
Mentions: Single mutants of the vWA domain cysteines did not drastically affect the migration pattern, i.e. both the precursor and the mature form were observed (Fig 4A), indicating that a significant proportion of the protein was able to exit the ER. Immunofluorescence analysis and surface biotinylation experiments further showed that mutation of Cys-39 or Cys-218 to alanine does not significantly affect targeting of CMG2 to the cell surface (Fig 4B and E).

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