Regulation and Mechanisms of ER Proteostasis

ABSTRACT The endoplasmic reticulum (ER) plays a pivotal role in maintaining protein homeostasis by implementing crucial quality control mechanisms. Newly synthesized polypeptides that fail to fold correctly, are directed for degradation by the proteasome through ER-associated degradation (ERAD) or transported to the lysosome for

degradation via autophagic processes known as ER-autophagy (ER-phagy). Although ERAD and ER-phagy share common features, these pathways function independently to recognize and degrade substrates. Despite the critical role of ER quality control, there are gaps in our understanding of this process including a) how

damaged proteins are directed into specific proteostasis pathways and b) the role of cell-type specific proteostasis adaptations. To address these gaps, the overall objective of this application is to understand how ER quality control is regulated in different cell types. Here we outline three goals (1) Establish the principles

governing the selection of targets by the ERAD and ER-phagy system; (2) Define how glycan site occupancy regulates the selection of ER quality control pathways; (3) Understand the cell-type specific roles of ER-phagy receptors. We previously found that a disease-causing mutation in the NPC1 protein, an isoleucine-to-

threonine substitution mutant at position 1061 (I1061T), is rapidly cleared from the ER by both ERAD and ER- phagy, making I1061T-NPC1 an excellent substrate to study this selection process. Additionally, we found that differences in glycan site occupancy between species alter NPC1 ER degradation. To accomplish the goals,

we will leverage our recently developed induced pluripotent stem cells (iPSCs) expressing dCas9, and/or NPC1 variants: WT-NPC1, I1061T-NPC1, or NPC1-null. This isogenic cell system enables the differentiation of iPSCs into hepatocytes or excitatory neurons and modulation of cell type-specific proteostasis through small

molecules or by stably reducing specific gene products using dCas9. In goal (1) we will use small molecule inhibitors and dCAS9 to study the role of glycan structures in ERAD and ER-phagy pathway selection. Goal (2) involves introducing human and mouse NPC1 glycan mutants into NPC1-null iPSCs using lentivirus. These

cells will be differentiated into neurons and hepatocytes to study the role of glycan sites in NPC1 degradation using biochemical methods. Lastly, in goal (3) we will use dCas9 to stably reduce the expression of a panel of ER-phagy receptors in iPSC derived neurons and hepatocytes. Then we will leverage LC-MS/MS and

biochemical methods to study their role in regulating the cell-type specific proteome. The rationale for this project is that understanding the recognition and evasion processes in ER quality control has significant implications for many human disorders, including Cystic Fibrosis (CFTR), alpha-1 antitrypsin deficiency (ATZ),

and Gaucher disease (GCase). These insights could potentially lead to novel treatments for these conditions.