Alzheimer disease (AD) is a neurodegenerative disorder characterized by a decline in thinking and independence in personal daily activities, and usually affects people over the age of 65. In China, the prevalence of Alzheimer's disease (AD) increased with the aging population (Ren, Rujing et al, 2022). AD's occurrence, impact, and fatality rates have consistently risen, making it the fifth leading cause of death among both urban and rural residents in China.

Consequently, this has caused substantial financial burdens on individuals, families, and society. According to a national study, simulated Chinese AD prevalence during 2011-2050 from 6-28 million. the annual cost of treating AD patients in China was US$167.74 billion in 2015, and this study is projected to reach US$1.8 trillion by 2050 due to continually rising treatment costs (CAWA, 2020). The lack of medical specialists and limited public awareness both triggered social and health problems. As a result, there is an urgent need to improve the prevention and treatment of AD.

α2A-adrenoceptor (α2A-AR) induces Alzheimer’s Disease (AD) because of its overdistribution across the cell membrane. (WHY and HOW）Therefore, our goal is to limit the amount of α2A-AR on cell membranes so it does not overload. α2A-AR alone will not cause AD if the is within the cell while not attached to the membrane. α2A-adrenoceptor, being a membrane protein, requires transport proteins to move to the cell membrane after transcription and translation. So our group shifted our target to its transport proteins.

 

With the inspiration of Dr. Xu’s work (Xu X. et al, 2022) and her support, we confirmed our experimental design that is to determine which transport protein is responsible for the conveyancing of α2A-AR and lower the production of such protein. This is done through 3 parts: protein purification, IP-MS and RUSH-BRET.

 

Protein Purification 

The first step is to construct α2A-AR protein with GST-tag in the C terminal and then purify it to a protein that also contains a GST tag. 

 

IP-MS

Immunoprecipitation-mass spectrometry (IP-MS) and DNA sequencing could be considered to identify which specific proteins could be the candidates to potentially possess the ability to effect the transportation of α2A-AR to cell membranes.

 

RUSH-BRET

After determining our candidate transport proteins, we need a way to measure the level of α2A-AR within a cell. The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor. Moreover, BRET can also be used as an indicator of subcellular location. 

The Renilla luciferase Rluc8 was fused to the C-terminus of the α2AR (α2AR-Rluc8) to serve as a BRET donor. The fluorescent protein venus was fused to the N-terminus of a C-terminal fragment of KRas (V-kras) to serve as a plasma membrane-associated BRET acceptor. BRET donor and BRET acceptor interaction produced a substantial BRET signal.

 

Diagram of α2AR (α2AR-Rluc8) transportation to the cell membrane. Image By Xu XING

 

KDEL is connected to Streptavidin (Str), this complex was fused to the SBP-α2AR -Rluc8, KEDEL can help Str- SBP-α2AR to localize to endoplasmic reticulum (ER).

Biotin can bind Str to disturb KDEL and SBP-α2AR -Rluc8 interaction, SBP-α2AR -Rluc8 will be released and transform from ER to cell membrane. On the cell membrane, Energy is transferred when Rluc8 of SBP-α2AR -Rluc8 replace the RAS of the Venus-K-RAS on the cell membrane. So we can use RUSH-BRET technique to detect the content of SBP-α2A-AR on the cell membrane and identify which target that can effectively decrease the amount of α2A-AR on the cell membrane.

 

 

1. The plasmid GST-α2A-AR-CTand Str-KDEL_SBP-α2A-AR- Rluc8 are successfully constructed, which are identified by sequencing.

2. Pure GST-α2A-AR-CT protein products with biological functions could be obtained from E. coli BL21. SDS-PAGE would be applied to validate the purification..

3. The pure GST-α2A-AR-CT protein would be used for IP-MS and several candidate proteins could be found for further research.

4. Str-KDEL_SBP-α2A-AR- Rluc8 can be expressed in HEK293 cell, add biotin can promote α2A-AR move to cell membrane from ER. RUSH-BRET platform can be used to detect the content of α2A-AR on the cell membrane.

 

 

[1]  Ren, Rujing, et al. "The China Alzheimer Report 2022." General Psychiatry 35.1 (2022).

[2]  Chinese Aging Well Association. Alzheimer’s Disease Chinese . Survey on the living status of Alzheimer's disease patients in China of 2019. (2020) http://weixin.moreedge.cn/bps_2019/index.php

[3]  Xu, X. and Wu, G., 2022. Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport. Journal of Biological Chemistry, 298(6).