Investigation of lysosomal phosphoproteome changes in altered cholesterol metabolism in Niemann-Pick Disease Type C (NPC)

Abstract

The hypothesis underlying this thesis is that the function of lysosomal membrane proteins can be regulated by interconversion, specifically phosphorylation. This was investigated in a drug induced cellular model of the lysosomal storage disorder Niemann-Pick disease Type C (NPC). In this disease mutations in genes of two distinct proteins, NPC1 or NPC2, cause impaired cholesterol efflux from late endosomes/lysosomes to the endoplasmic reticulum. This results in the accumulation of unesterified cholesterol inside the lysosome and it was hypothesized that this causes changes in the lysosomal phosphoproteome. To that context, lysosomal cholesterol storage was induced in mouse embryonic fibroblasts by U18666A, an inhibitor of NPC1. <br /> The phosphoproteome of lysosomes enriched from untreated and U18666A treated cells was compared to identify quantitative changes of protein phosphorylation. Mass spectrometry-based phosphoproteomic analysis of lysosomes identified and quantified a total of 7627 phosphosites with an upregulation of 123 phosphosites on U18666A treated samples. Of these, 11 phosphopeptides could be assigned to lysosomal proteins proving that in fact lysosomal proteins can be differentially phosphorylated. In an effort to demonstrate functional consequences of phosphorylation one of the 11 proteins - heparan α-glucosaminide N-acetyltransferase (HGSNAT) essential for heparan sulfate (HS) degradation - was chosen for more detailed examination. The 2.36-fold increased phosphorylation of serine211 within the cytosolic domain of this enzyme correlated well with a 2.8-fold increase of activity upon U18666A treatment suggesting that phosphorylation regulated HGSNAT activity. Moreover, blocking heparan sulfate degradation by suramin treatment decreased HGSNAT activity 4-fold, indicating that HS catabolism influences HGSNAT activity. However, phosphomimetic and phosphoresistant amino acid substitutions of serine211 did not show any significant change in HGSNAT activity ruling out that phosphorylation regulates activity. Moreover, immunohistochemistry showed that wild type and all HGSNAT mutants, localize to lysosomes, indicating that phosphorylation has no effect on subcellular localization of HGSNAT. When enriched lysosomes were treated with shrimp alkaline phosphatase, surprisingly dephosphorylation caused a ~2.5-fold increase of HGSNAT activity. This was only observed with endogenous HGSNAT in non-overexpressing cells. When lysosomes enriched from cells overexpressing wild type or serine211 phosphomimetic/resistant HGSNAT, dephosphorylation had no effect. Since this suggested that HGSNAT interacting proteins influencing its activity may exist, SILAC (Stable isotope labeling of amino acids in cell culture)-based co-immunoprecipitation experiments to identify HGSNAT interacting proteins were performed.