RNF13 is a novel interactor of iduronate 2-sulfatase that modifies its glycosylation and maturation
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Abstract
Mucopolysaccharidosis type II, also known as Hunter syndrome, is a rare and fatal disease caused by mutations in the iduronate 2-sulfatase (IDS) encoding gene. The enzymatically inactive variant proteins lead to pathological accumulation of glycosaminoglycans in the lysosomes, causing dysfunction in multiple organs. IDS is expressed as a precursor protein, and its processing and lysosomal targeting are crucial for proper enzymatic activity. However, IDS intracellular dynamic is poorly understood and a better understanding of its processing mechanisms would benefit the development of new therapeutic strategies. AlphaFold 3 predicted an interaction between IDS and the E3 ubiquitin ligase RNF13. Co-immunoprecipitation assays confirm this interaction and further show that RNF13 interacts preferentially with a predominantly underglycosylated immature form of IDS, resulting in altered IDS glycosylation and maturation. The results demonstrate that IDS glycosylation site Asn246 is important for lysosomal targeting, although its glycosylation is not altered by RNF13. This study also unravels that RNF13 forms a heterodimer with the E3 ubiquitin ligase RNF167 that modify both RNF13 and RNF167 lysosomal trafficking. In addition, the heterodimer interacts and alters IDS differently than RNF13 or RNF167 alone. RNF13 catalytic E3 ligase activity is required to generate an underglycosylated form, but not that of RNF167. This study exposes that the proteasome rapidly degrades IDS underglycosylated forms, and RNF13 exerts a protective effect. Overall, this study reveals a novel and dual role of RNF13 on IDS maturation and degradation, providing mechanistic insights into IDS trafficking.
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This study highlights the true complexity of protein processing, trafficking, and its ultimate function! I think it could benefit from some orthogonal methods of analysis - would be it be possible to try to isolate specific 'states' of processed protein (like you did with the CHX treatment) or using subcellular localization or IPs to identify exactly which glycosylation sites or cleavages have been made? Excited to see what comes next!
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with Lamp1
Some Lamp1 staining for lysosome localization could contribute to the above IF figures as well!
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glycosylated residue.
I wonder if there might be a more straightforward way to probe this interaction. It seems difficult to study the interaction between RNF13 and IDS because there are so many different precursors or processed forms of IDS. Perhaps a strategic approach could be to either isolate the process (in cells) for which the interaction is most important, and then determine which processed form of IDS is involved. Or maybe purify different forms of IDS and determine using binding assays, since IPs are less capable of identifying 'direct interactions'.
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proteins to mature
This is a clever way to probe protein processing! It might be useful to do some western blotting with this treatment to look at the impact on processing that way as well.
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omplex
What is your hypothesis for this interaction, then? Does RNF13 interact with both species, throughout the whole processing stage? Or predominantly with one species?
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and maturation.
This is a very neat result! I'm wondering if it might be interesting to repeat an IP with different fractions - like a lysosome-enriched fraction vs a whole lysate. There are some pretty crude lysosome preps that could give some nice connections between forms that are trafficked, the maturation data you have, and the localization data you have below.
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