Regulation and mechanisms of ER proteostasis

Active Project Grant Awarded Last action

Award ID
R35GM154959
Recipient
Awarding agency
Obligated
$1,525,004

Award facts

Assistance listing
93.859 Biomedical Research and Research Training
Completion date 45% complete
Place of performance
Iowa City, IA 52242, USA

Award details come from USAspending.gov.

Description

As the agency reported it.

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.

Transactions

Action Obligated Date
Amendment $381,251
Amendment $381,251
Amendment $381,251
Original award $381,251

Quick answers

Who received award R35GM154959?

R35GM154959 was awarded to The University Of Iowa by National Institute of General Medical Sciences (National Institutes of Health) on August 1, 2024.

How much is award R35GM154959 worth?

$1,525,004 has been obligated on R35GM154959.

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