# Defining the nuclear envelope-resident protein turnover machinery implicated in diseases affecting cholesterol metabolism

> **NIH NIH R01** · YALE UNIVERSITY · 2021 · $309,370

## Abstract

Project Summary/Abstract
 The Lamin B receptor (LBR) is a polytopic membrane protein residing at the nuclear envelope in close
association with the nuclear lamina. We discovered that human LBR has an essential role in cholesterol
synthesis, which is strongly perturbed by LBR mutations responsible for the congenital disorders Greenberg
Dysplasia and Pelger-Huët anomaly. Several of these disease-associated mutations lead to the rapid
degradation of LBR at the inner nuclear membrane (INM), a site that is poorly understood from the perspective
of protein quality control. Progress in this area has been hampered by the absence of suitable methodologies,
with the lack of model substrates being the major limitation. This deficiency in our understanding of cellular
quality control presents a major obstacle towards understanding and treating congenital diseases caused by
mutations in nuclear envelope-resident proteins, commonly referred to as nuclear envelopathies.
 A detailed characterization of the protein turnover mechanisms responsible for the elimination of LBR
disease mutants has allowed us to directly observe inner nuclear membrane protein turnover (INMPT) in
mammalian cells for the first time. We have since established LBR-based model substrates and readouts,
allowing us to initiate an unprecedented interrogation of protein turnover mechanisms in the nuclear envelope.
As a logical extension of these endeavors, we now propose to exploit our newly established experimental
platform to identify the machinery responsible for protein quality control at the INM. Two independent yet
synergistic approaches will be pursued to achieve this goal: (1) Based on our established strategies to
intersect the degradation of LBR derivatives at distinct stages of membrane extraction and proteasomal
degradation, we will isolate stalled degradation intermediates to identify associated factors involved in INMPT
via mass spectrometry; (2) We will conduct an unbiased, genome-wide CRISPR/Cas9-based screen to
discover genes implicated in INMPT. To this end, we will utilize a cell line expressing a degradation-prone,
split-GFP derivative of LBR. Mutant stabilization upon functional inactivation of a gene required for INMPT will
lead to an increase in fluorescence, allowing us to identify the targeted gene via FACS analysis and next-
generation sequencing. For candidates arising from both approaches (1) and (2), we will (3) functionally
characterize these factors using our firmly established experimental platform including (i) readouts for
localization to the nuclear envelope; (ii) assays for membrane dislocation/extraction and (iii) ubiquitination and
proteasomal degradation, thus allowing us to assign a function to each validated candidate gene or interacting
protein. The results of these studies will allow us to define the protein homeostasis network that safeguards
protein integrity at the nuclear envelope. Apart from closing a major gap in our understanding of cellular pro...

## Key facts

- **NIH application ID:** 10063878
- **Project number:** 5R01GM126835-04
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Christian Dirk Schlieker
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $309,370
- **Award type:** 5
- **Project period:** 2018-01-01 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10063878

## Citation

> US National Institutes of Health, RePORTER application 10063878, Defining the nuclear envelope-resident protein turnover machinery implicated in diseases affecting cholesterol metabolism (5R01GM126835-04). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10063878. Licensed CC0.

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