Abstract
Niemann–Pick type C disease is a fatal, progressive neurodegenerative disorder caused by loss-of-function mutations in NPC1, a multipass transmembrane glycoprotein essential for intracellular lipid trafficking. We sought to define the cellular machinery controlling degradation of the most common disease-causing mutant, I1061T NPC1. We show that this mutant is degraded, in part, by the proteasome following MARCH6-dependent ERAD. Unexpectedly, we demonstrate that I1061T NPC1 is also degraded by a recently described autophagic pathway called selective ER autophagy (ER-phagy). We establish the importance of ER-phagy both in vitro and in vivo, and identify I1061T as a misfolded endogenous substrate for this FAM134B-dependent process. Subcellular fractionation of I1061T Npc1 mouse tissues and analysis of human samples show alterations of key components of ER-phagy, including FAM134B. Our data establish that I1061T NPC1 is recognized in the ER and degraded by two different pathways that function in a complementary fashion to regulate protein turnover.
Highlights
Niemann–Pick type C disease is a fatal, progressive neurodegenerative disorder caused by loss-of-function mutations in NPC1, a multipass transmembrane glycoprotein essential for intracellular lipid trafficking
I1061T NPC1 accumulates after lysosomal or proteasomal I1061T NPC1 is degraded in part by MARCH6-dependent inhibition
We demonstrate that I1061T NPC1 is degraded by two complementary pathways that target the misfolded, mutant protein to either the proteasome or autophagosome (Fig. 8d)
Summary
Niemann–Pick type C disease is a fatal, progressive neurodegenerative disorder caused by loss-of-function mutations in NPC1, a multipass transmembrane glycoprotein essential for intracellular lipid trafficking. To begin defining the mechanisms of I1061T degradation, we analyzed lysates from control (CTRL) cells expressing WT NPC1 and I1061T homozygous primary fibroblasts after treatment with the proteasome inhibitors MG132 or epoxomicin (Epox) at non-toxic concentrations (Supplementary Fig. 1a). WT NPC1 protein levels significantly increased following treatment with Baf (Fig. 1a), consistent with its stabilization within the lysosome.
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