PXD005357

PXD005357 is an original dataset announced via ProteomeXchange.

Title	Cystinosin turnover
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.
HostingRepository	PRIDE
AnnounceDate	2017-01-16
AnnouncementXML	Submission_2017-01-16_08:58:16.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Chiara guerrera
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2016-11-15 05:50:05	ID requested
⏵ 1	2017-01-16 08:58:18	announced

Nevo N, Thomas L, Chhuon C, Andrzejewska Z, Lipecka J, Guillonneau F, Bailleux A, Edelman A, Antignac C, Guerrera IC, Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Mol Cell Proteomics, 16(3):457-468(2017) [pubmed]

curator keyword: Biomedical

submitter keyword: cystinosin, cystinosis, ER retention, dynamic SILAC, turnover

Ida Chiara Guerrera
contact affiliation	Plateforme Protéomique Paris Descartes Necker, PPN, 3P5-Necker, SFR Necker, US24, 75014 Paris, France
contact email	chiara.guerrera@inserm.fr
lab head
Chiara guerrera
contact affiliation	Faculte de Medecine
contact email	chiara.guerrera@inserm.fr
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD005357
     1. Label: PRIDE project
     2. Name: Cystinosin turnover