Niemann-Pick disease, type C (NPC1) is a fatal autosomal recessive, neurodegenerative, lysosomal storage disorder, caused by mutations in the NPC1 gene with no FDA-approved therapy. As a result, lysosomal accumulation of unesterified cholesterol and glycosphingolipids are observed. While much effort has been focused on understanding the mechanisms contributing to the pathophysiology of the disease, much less has been revealed about the NPC1 protein itself beyond a role in cholesterol transport. Here, we aimed to investigate protein-protein interactions of NPC1 to provide a deeper understanding of the function of this protein beyond lipid transport. We pursued this question by co-immunoprecipitating (Co-IP) NPC1 and interacting partners in two different systems. First, cortical tissues from wild-type Balb/c Npc1nih (NPC1-null) mice as well as NPC1-null animals as negative controls for non-specific binding. Second, we examined interactions involving NPC1 when the protein is non-functional but expressed in the HT-22 mouse neuronal cell line and U18666A, a NPC1 inhibitor. In both studies, Co-IP eluents were digested using the S-Trap method and peptides were separated and analyzed with an Agilent 1260 nLC coupled with a Thermo QExactive mass spectrometer. Proteins were identified using Proteome Discoverer equipped with the SEQUEST search engine. We observed 65 potential interactors of NPC1 in cortical tissues, whereas 321 proteins interacted with NPC1 despite inhibiting activity in HT-22 cells. Protein clusters related to ribosomal proteins and actin-related complex were found to be consistent in both cortical and drug-treated experiments. The Arp2/3 complex was identified in both experiments and plays a crucial role in many cellular processes including regulation of the cytoskeletal network. Thus, we sought to further understand the interaction between Arp2/3 complex and NPC1 through mapping the distribution of this complex and actin network in primary cortical neurons. Together, we have used multiple model systems to reveal a new interaction, NPC1-Arp2/3, and are currently investigating functional aspects of this new interaction.

Mass spectrometry protein interactome study reveals new roles for the classical cholesterol transporter, NPC1

ACS Spring 2022

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