# Regulation and Mechanisms of ER Proteostasis

> **NIH NIH R35** · UNIVERSITY OF IOWA · 2024 · $381,251

## Abstract

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 c...

## Key facts

- **NIH application ID:** 10937803
- **Project number:** 1R35GM154959-01
- **Recipient organization:** UNIVERSITY OF IOWA
- **Principal Investigator:** Mark Louis Schultz
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $381,251
- **Award type:** 1
- **Project period:** 2024-08-01 → 2029-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10937803

## Citation

> US National Institutes of Health, RePORTER application 10937803, Regulation and Mechanisms of ER Proteostasis (1R35GM154959-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10937803. Licensed CC0.

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