DGIST Scholar: XBP1s negatively regulates LXRα and cholesterol efflux through an E3 ligase RNF181

Department of New Biology Theses Ph.D.

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XBP1s negatively regulates LXRα and cholesterol efflux through an E3 ligase RNF181

Title: XBP1s negatively regulates LXRα and cholesterol efflux through an E3 ligase RNF181

Author(s): Hyuncheol Ro

DGIST Authors: Hyuncheol Ro ; Jaemin Lee ; Byung-hoon Lee

Advisor: 이재민

Co-Advisor(s): Byung-hoon Lee

Issued Date: 2024

Awarded Date: 2024-02-01

Type: Thesis

Description: Macrophage; Atherosclerosis; LXRα; XBP1s; RNF181

Table Of Contents: Ⅰ. Introduction 1
1.1 Macrophage and Cholesterol efflux 1
1.2 LXR and cholesterol Metabolism 3
1.3 XBP1s and ER stress 6
1.4 RNF181 and E3 ligase 10
1.5 Aims of the Research 12
1.5.1 Aim1: Examine the detailed mechanism of decreased LXRα activity by XBP1s 12
1.5.2 Aim2: Identify a specific mediator of the mechanism behind the decreased LXRα activity by XBP1s 13
1.5.3 Aim3: Assessing the impact of decreased LXRα activity on cholesterol efflux in macrophage cells 13
Ⅱ. Materials and Methods 15
2.1 Mice and Diets 15
2.2 Reagents and plasmids 15
2.3 Cell Culture 16
2.4 Western blot 17
2.5 Luciferase assay 18
2.6 Co-immunoprecipitation and ubiquitination assay 18
2.7 Cycloheximide assay 19
2.8 qRT-PCR 19
2.9 Yeast two-hybrid 20
2.10 Adenovirus 21
2.11 Cholesterol efflux assa 21
2.12 Serum cholesterol quantification 22
2.13 Bone marrow derived macrophage (BMDM) preparation 22
2.14 Primers, siRNA and shRNA 23
2.15 Public CHIP-seq Data Analysis 26
2.16 Statistical Analysis 26
Ⅲ. Results 28
3.1 XBP1s reduce the LXRα stability by promoting ubiquitination 28
3.2 XBP1s Promotes the Expression of RNF181, a potential E3 Ligase Targeting LXRα 37
3.3 RNF181 is the E3 ligase that targets LXRα 51
3.4 RNF181 modulates endogenous LXRα and cholesterol efflux in human macrophage cell line 59
3.5 RNF181 affects in vivo macrophage LXRα and whole-body metabolism 68
Ⅳ. Conclusions and Discussion 72
4.1 Conclusion of Aim1 72
4.2 Conclusion of Aim2 72
4.3 Conclusion of Aim3 73
4.4 Discussion 73
Ⅴ. References 76
Ⅵ. 국문 요약문 83