Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. ApoE is a polymorphic protein with three major isoforms, apoE2, apoE3 and apoE4. The apoE isoforms differ from one another only by a single amino acid substitution, yet they have profound functional consequences at both the cellular and molecular levels. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural studies of full-length apoE and the apoE C-terminal domain is hindered by apoE's oligomerization property. It is well established that the C-terminal domain causes apoE aggregation. A monomeric, biologically active apoE C-terminal domain has been generated in our laboratory recently, which has solved the major technical problem in the apoE structural study. The NMR spectra of this monomeric, biologically active apoE C-terminal domain has been completely assigned and its NMR structure will be solved soon. This progress places us in a very good position to propose a NMR structural determination of full-length apoE. This research proposal focuses on solving the NMR structure of full-length human apoE in the lipid-free state using nuclear magnetic resonance (NMR) and molecular biology techniques. In addition, a high-level expression and refolding system has been established for the LDL receptor ligand-binding domain repeats (LDLR-LBDR) and LRP ligand binding domain 2 repeats (LRP-LBD2R), allowing us to propose to characterize the structural changes of the binding residues in apoE upon interaction with receptors. Finally, identification of the critical residues that are involved in the apoE domain-domain interactions has also been proposed in this proposal. Due to the importance of apoE and the apoE/receptor interactions in several major human diseases, including atherosclerosis and Alzheimer's disease, the significance of this proposal is very well justified. It is worth noting that one unique feature of this application is that several independent approaches have been proposed for each specific objective, ensuring that any success in one approach will achieve the overall goal of this objective. ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL074365-03
Application #
6915136
Study Section
Metabolism Study Section (MET)
Program Officer
Applebaum-Bowden, Deborah
Project Start
2003-07-01
Project End
2007-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
3
Fiscal Year
2005
Total Cost
$263,594
Indirect Cost
Name
Wayne State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Murray, Victoria; Huang, Yuefei; Chen, Jianglei et al. (2012) A novel bacterial expression method with optimized parameters for very high yield production of triple-labeled proteins. Methods Mol Biol 831:1-18
Chen, Jianglei; Wang, Jianjun (2011) A segmental labeling strategy for unambiguous determination of domain-domain interactions of large multi-domain proteins. J Biomol NMR 50:403-10
Chen, Jianglei; Li, Qianqian; Wang, Jianjun (2011) Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions. Proc Natl Acad Sci U S A 108:14813-8
Chen, Jianglei; Liu, Chia-Chen; Li, Qianqian et al. (2011) Two structural and functional domains of MESD required for proper folding and trafficking of LRP5/6. Structure 19:313-23
Chen, Jianglei; Li, Qianqian; Liu, Chia-Chen et al. (2010) NMR structure note: solution structure of the core domain of MESD that is essential for proper folding of LRP5/6. J Biomol NMR 47:283-8
Sivashanmugam, Arun; Wang, Jianjun (2009) A unified scheme for initiation and conformational adaptation of human apolipoprotein E N-terminal domain upon lipoprotein binding and for receptor binding activity. J Biol Chem 284:14657-66
Zhang, Yonghong; Chen, Jianglei; Wang, Jianjun (2008) A complete backbone spectral assignment of lipid-free human apolipoprotein E (apoE). Biomol NMR Assign 2:207-10
Zhao, Wentao; Zhang, Yonghong; Cui, Chunxian et al. (2008) An efficient on-column expressed protein ligation strategy: application to segmental triple labeling of human apolipoprotein E3. Protein Sci 17:736-47
Li, Qianqian; Huang, Yuefei; Xiao, Nan et al. (2008) Real time investigation of protein folding, structure, and dynamics in living cells. Methods Cell Biol 90:287-325
Chen, Jianglei; Bu, Guojun; Wang, Jianjun (2007) A complete NMR spectral assignment of the conserved region of the MESD protein, MESD(12-155). Biomol NMR Assign 1:3-5

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