Proprotein convertase subtilisin kexin type 9 (PCSK9) regulates plasma cholesterol levels by enhancing the intracellular degradation of the LDL receptor (LDLR), the primary driver of LDL clearance from plasma. Human PCSK9 is composed of 692 amino-acids. After removal of the signal peptide (residues 1-30), the secreted PCSK9 presents three domains: 1. The N-terminal pro-domain (PD, residues 31-152), which is cleaved but remains associated with the rest of the protein, and displays a highly disordered N-terminus (residues 31-60);2. The catalytic domain (CA, residues 153-449), which contains a proteolytic site masked and neutralized by the associated PD;3. The C-terminal domain (CD, residues 453-692), which consists of three tightly packed modules, CM1 (residues 457-527), CM2 (residues 534-601) and CM3 (residues 608-679). The consensus view is that PCSK9 binds to the epidermal growth factor type A (EGF-A) repeat of the LDLR via its CA, but does not cause direct proteolysis of the LDLR. Understanding the function of PSCK9 is central to the development of novel therapeutic agents to reduce cholesterol levels and cardiovascular risk. Although PCSK9 primarily targets the hepatic LDLR, it may also function on other lipoprotein receptors, such as apoER2, LRP1, and VLDLR, as well as on LDLR in extra-hepatic tissues. Our published work and preliminary studies have addressed the role of different PCSK9 domains in the secretion and function of the protein. We have found that: 1. Deletion of CM2 produces a protein that is secreted and functional;2. Deletion of either CM1 or CM3 produces a protein that is neither secreted nor functional;3. Deletion of the entire CD produces a protein that is secreted but not functional;4. The PD of a PCSK9 fragment lacking CD facilitates the secretion of a PCSK9 fragment lacking PD;5. The discrete CD of PCSK9 binds the ectodomain of the LDLR in solution and dose- dependently inhibits the LDLR-degrading effect of full-length PCSK9 cells;6. PCSK9 undergoes pH-dependent self-association, which is mediated by the CA and increases the LDLR-reducing functionality;7. Over- expression of PCSK9 reduces LDLR, apoER2, and LRP1 in HEK293T cells, and reduces LDLR levels in macrophages;8. Transgenic expression of human PCSK9 in macrophages reduces liver LDLR and raises plasma cholesterol levels in mice. The central hypothesis of this proposal is that PCSK9 uses multiple domain interactions for secretion and function, and contributes to atherogenesis by targeting multiple lipoprotein receptors. We plan to: 1. Examine the role of the C-terminal domain in PCSK9 secretion and LDLR binding;2. Test the hypothesis that domain interaction and self-association modulate PCSK9 secretion and function;3. Test the hypothesis that PCSK9 secreted by macrophages modifies the biology of the atheroma through multiple effects on lipoprotein receptors. Investigating these novel roles of PCSK9's C-terminal domain and pro-domain may provide new leads in the development of inhibitors and advance our understanding of plasma cholesterol regulation.

Public Health Relevance

PCSK9 is a newly identified protein that regulates plasma cholesterol levels in humans, thereby determining individual predisposition to coronary artery disease. By producing mutant forms of this protein, we have obtained preliminary data suggesting that there are specific structural requirements for the secretion and function of PCSK9. We are proposing to investigate these correlations in detail and to evaluate the overall impact of PCSK9 expression on lipoprotein receptors and atherosclerosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL106845-04
Application #
8606492
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Hasan, Ahmed a K
Project Start
2011-04-01
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
4
Fiscal Year
2014
Total Cost
$944,117
Indirect Cost
$237,273
Name
Vanderbilt University Medical Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Tavori, Hagai; Christian, Devon; Minnier, Jessica et al. (2016) PCSK9 Association With Lipoprotein(a). Circ Res 119:29-35
Giunzioni, Ilaria; Tavori, Hagai; Covarrubias, Roman et al. (2016) Local effects of human PCSK9 on the atherosclerotic lesion. J Pathol 238:52-62
Shapiro, Michael D; Fazio, Sergio (2016) From Lipids to Inflammation: New Approaches to Reducing Atherosclerotic Risk. Circ Res 118:732-49
Tavori, Hagai; Giunzioni, Ilaria; Predazzi, Irene M et al. (2016) Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms. Cardiovasc Res 110:268-78
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Tavori, Hagai; Rashid, Shirya; Fazio, Sergio (2015) On the function and homeostasis of PCSK9: reciprocal interaction with LDLR and additional lipid effects. Atherosclerosis 238:264-70
Rashid, Shirya; Tavori, Hagai; Brown, Patrick E et al. (2015) Response to letter regarding article, ""Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and -independent mechanisms Circulation 131:e428
Shapiro, Michael D; Fazio, Sergio; Tavori, Hagai (2015) Targeting PCSK9 for therapeutic gains. Curr Atheroscler Rep 17:499
Jahangir, Eiman; Lipworth, Loren; Edwards, Todd L et al. (2015) Smoking, sex, risk factors and abdominal aortic aneurysms: a prospective study of 18?782 persons aged above 65 years in the Southern Community Cohort Study. J Epidemiol Community Health 69:481-8

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