Dataset Information

Cystinosin interactomics - Impact of cystinosin glycosylation on protein stability by differential dynamic SILAC

ABSTRACT: Cystinosis is a rare autosomal recessive lysosomal storage disorder, characterized by an intra-lysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations are reported, leading to different severity of the disease, often in correlation with cystinosin residual activity as a transporter and with maintenance of its protein-protein interactions. In this study we focus on ΔITILELP the only mutation reported, in some cases, to lead to severe forms, inconsistently with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the 7 glycosylation sites of the protein. Our hypothesis is that ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic SILAC study clearly shows that wild-type cystinosin is very stable while ΔITILELP is degraded three times faster than the wild-type protein. Additional lysosome inhibition experiments confirm ΔITILELP instability and show that the degradation is mainly lysosomal. We can observe that at the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway like the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies show that ΔITILELP is partially retained at the ER. We propose that ΔITILELP mutation causes protein misfolding, ER retention and incapability to be processed in the Golgi, as we demonstrate that ΔITILELP carries high mannose glycans on all its 6 remaining glycosylation sites. Altogether, we show that the high turnover of ITILELP, due to its immature glycosylation state together with low transport activity, might be responsible for the phenotype observed in some patients.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Chiara guerrera

LAB HEAD: Ida Chiara Guerrera

PROVIDER: PXD004948 | Pride | 2017-01-16

REPOSITORIES: Pride

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Publications

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).

Nevo Nathalie N Thomas Lucie L Chhuon Cerina C Andrzejewska Zuzanna Z Lipecka Joanna J Guillonneau François F Bailleux Anne A Edelman Aleksander A Antignac Corinne C Guerrera Ida Chiara IC

Molecular & cellular proteomics : MCP 20170112 3

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is <i>CTNS</i>, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔIT ...[more]

PMID: 28082515

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Project description:Cystinosis is a rare autosomal recessive lysosomal storage disorder, characterized by an intra-Cystinosis is a rare autosomal recessive lysosomal storage disorder, characterized by an intra-lysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations are reported, leading to different severity of the disease, often in correlation with cystinosin residual activity as a transporter and with maintenance of its protein-protein interactions. In this study we focus on ΔITILELP the only mutation reported, in some cases, to lead to severe forms, inconsistently with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the 7 glycosylation sites of the protein. Our hypothesis is that ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic SILAC study clearly shows that wild-type cystinosin is very stable while ΔITILELP is degraded three times faster than the wild-type protein. Additional lysosome inhibition experiments confirm ΔITILELP instability and show that the degradation is mainly lysosomal. We can observe that at the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway like the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies show that ΔITILELP is partially retained at the ER. We propose that ΔITILELP mutation causes protein misfolding, ER retention and incapability to be processed in the Golgi, as we demonstrate that ΔITILELP carries high mannose glycans on all its 6 remaining glycosylation sites. Altogether, we show that the high turnover of ITILELP, due to its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients. lysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations are reported, leading to different severity of the disease, often in correlation with cystinosin residual activity as a transporter and with maintenance of its protein-protein interactions. In this study we focus on ΔITILELP the only mutation reported, in some cases, to lead to severe forms, inconsistently with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the 7 glycosylation sites of the protein. Our hypothesis is that ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic SILAC study clearly shows that wild-type cystinosin is very stable while ΔITILELP is degraded three times faster than the wild-type protein. Additional lysosome inhibition experiments confirm ΔITILELP instability and show that the degradation is mainly lysosomal. We can observe that at the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway like the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies show that ΔITILELP is partially retained at the ER. We propose that ΔITILELP mutation causes protein misfolding, ER retention and incapability to be processed in the Golgi, as we demonstrate that ΔITILELP carries high mannose glycans on all its 6 remaining glycosylation sites. Altogether, we show that the high turnover of ITILELP, due to its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

Dataset Information

Cystinosin interactomics - Impact of cystinosin glycosylation on protein stability by differential dynamic SILAC

Publications

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).

Similar Datasets

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