Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell ABCA1

Cholesterol Content
3. Inhibition of Ubiquitination and Lysosomal Degradation of csABCA1 by LXRβ
4.
Decreased Interaction of ABCA1 With LXRβ and Increased Ubiquitination of
28
Discussion
29
Discussion
Cholesterol Accumulation
csABCA1 ubiquitination ESCRT lysosomal degradation
(csABCA1)
Promote the progression of atherosclerosis
Content
13
Results 2
Liver X receptor (LXR) agonist and acetylated low-density lipoprotein (acLDL) loading increased the amount of ubiquitinated ATP-binding cassette transporter A1 (ABCA1-Ub).
1
Background
2
Background
ApoA1
HDL
Ub Lysosomes
ABCA1
？
3
Objective
Investigated the relevance of the degradation pathway to the turnover of csABCA1 in hypercholesterolemia.
24
Results 4
High-fat diet ( HFD) feeding in mice lessened the interaction of ATP-binding cassette transporter A1 (ABCA1) with liver X receptor β (LXRβ) and facilitated the ubiquitination of ABCA1 in liver plasma membrane fractions (LPM).
30
Conclusion
31
Conclusion
Under conditions of high cellular cholesterol content, csABCA1 became
susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which
19
Results 3
bafilomycin
csABCA1
(bafilomycin completely blocked the accelerated degradation of csABCA1)
20
Results 3
Liver X receptor β (LXRβ) interacted with cell surface–resident ATP-binding cassette transporter A1 (csABCA1) and negatively modulated its ubiquitination.
Bafilomycin
csABCA1
(Bafilomycin canceled the degradation of csABCA1 facilitated)
15
Results 2
TSG101
csABCA1
(The accelerated degradation of csABCA1 was also blocked completely by the depletion of TSG101)
Tadahaya Mizuno, Hisamitsu Hayashi, Hiroyuki Kusuhara
Reporter
: Chong Huang (黄冲)
Supervisor : Chaoke Tang (唐朝克教授 ) May, 26, 2015
Institute of Cardiovascular disease, Key Laboratory for Arteriosclerology of Hunan Province, University of South China
16
Results 3
Inhibition of Ubiquitination and Lysosomal Degradation of csABCA1 by LXRβ
17
Results 3
Liver X receptor β (LXRβ) was associated with LXR agonist–mediated lysosomal degradation of cell surface–resident ATP-binding cassette transporter A1 (csABCA1) through its posttranscriptional function.
Involvement of Lysosomal Degradation Through the ESCRT Machinery in the Accelerated Degradation of csABCA1 Under Conditions of High Cellular Cholesterol
4
Methods
1. Reagents and antibodies 2. Animals, and peritoneal macrophage isolation
3. Cell culture
4. Short interfering RNA (siRNA) and Stealth RNAi (stRNA) 5. Construction of plasmid vectors
GW3965 (GW3965
csABCA1
accelerated degradation of csABCA1)
10
Results 1
GW3965 ＋ HppG2 HuH-7 MPM 30% － 56%
38%
58%
30%
61%
11
Results 1
acLDL
csABCA1
12
Results 2
GW3965 and acLDL
ABCA1-Ub
14
Results 2
Lysosomal degradation through endosomal sorting complex required for transport (ESCRT) pathway was involved in the accelerated degradation of cell surface–resident ATP-binding cassette transporter A1 (csABCA1) by liver X receptor (LXR) agonist.
6. Immunoprecipitation
7. Cell surface immunoprecipitation (csIP) 8. Cell surface biotinylation to evaluate the expression and degradation
rate of cell surface-resident protein
（COS1 cells）
21
Results 3
22
Results 3
FLAG–LXRβ
HA–ABCA1-Ub
(FLAG–LXRβ interacted with 207HA–ABCA1 and reduced the ubiquitination level of cs207HA–ABCA1.)
depletion of LXRβ
csABCA1
(Degradation of csABCA1 was facilitated by depletion of LXRβ)
18
Results 3
LXRβ inhibits the accelerated lysosomal degradation of csABCA1 by an LXR agonist
9. Immunoblotting 5
Results
6
Results
1. Accelerated Degradation of csABCA1 by Liver X Receptor Agonist and
Acetylated Low-Density Lipoprotein Loading
2. Involvement of Lysosomal Degradation Through the ESCRT Machinery in the Accelerated Degradation of csABCA1 Under Conditions of High Cellular
Arterioscler Thromb Vasc Biol. March 24, 2015 IF:5.3
Cellular Cholesterol Accumulation Facilitates Ubiquitination and
Lysosomal Degradation of Cell Surface–Resident ABCA1
23
Results 4
Decreased Interaction of ABCA1 With LXRβ and Increased Ubiquitination of ABCA1 in Liver Plasma Membrane Fractions of High-Fat Diet–Fed Mice