Dataset Information

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

ABSTRACT: Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, 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 ?ITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ?ITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ?ITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ?ITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ?ITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ?ITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ?ITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ?ITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ?ITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ?ITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

SUBMITTER: Nevo N

PROVIDER: S-EPMC5341006 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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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:Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models.

Dataset Information

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