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Quantitative Proteomic Analysis of PCSK9 Gain of Function in Human Hepatic HuH7 Cells

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Ottawa Institute of Systems Biology, University of Ottawa, Ontario, Canada
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada
§ Chronic Disease Program, Ottawa Hospital Research Institute, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada
*Drs. Daniel Figeys and Janice Mayne, Ottawa Institute of Systems Biology, University of Ottawa. DF e-mail: [email protected]. Tel: 613-562-5800 ext 8674. Fax 613-562-5655. JM e-mail: [email protected]. Tel: 613-562-5800 ext 8073. Fax 613-562-5655.
Cite this: J. Proteome Res. 2011, 10, 4, 2011–2026
Publication Date (Web):February 18, 2011
https://doi.org/10.1021/pr2000072
Copyright © 2011 American Chemical Society

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    Abstract

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    Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis, mediating degradation of the liver low-density lipoprotein receptor (LDLR). In fact, gain- and loss-of-function PCSK9 variations in human populations associate with hyper- or hypo- cholesterolemia, respectively. Exactly how PCSK9 promotes degradation of the LDLR, the identity of the other biomolecules involved in this process, and the global effect of PCSK9 on other proteins has not been thoroughly studied. Here we employ stable isotope labeling with amino acids in cell culture (SILAC) to present the first quantitative, subcellular proteomic study of proteins affected by the stable overexpression of a gain-of-function PCSK9 membrane-bound chimera (PCSK9-V5-ACE2) in comparison to control, empty vector transfections in a human hepatocyte (HuH7) cell line. The expression level of 327 of 5790 peptides was modified by PCSK9-V5-ACE2 overexpression. Immunoblotting was carried out for the control transferrin receptor, shown to be unaffected in cells overexpressing PCSK9-V5-ACE2, thus validating our SILAC results. We also used immunoblotting to confirm the novel SILAC results of up- and down-regulation of several proteins in cells overexpressing PCSK9-V5-ACE2. Moreover, we documented the novel down-regulation of the EH domain binding protein-1 (EHBP1) in a transgenic PCSK9 mouse model and its up-regulation in a PCSK9 knockout mouse model.

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    Supplementary Table 1: Total list of quantified proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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    2. Lei Huang, Ying Cheng, Yulian Mu, Kui Li, . PCSK9-D374Y Suppresses Hepatocyte Migration through Downregulating Free Cholesterol Efflux Rate and Activity of Extracellular Signal-Regulated Kinase. Analytical Cellular Pathology 2023, 2023 , 1-11. https://doi.org/10.1155/2023/6985808
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