Investigating the Role of PCSK9 C-terminal Domain in the Regulation of Low-Density Lipoprotein Receptor and Cholesterol Metabolism

• Author / Creator

  Deng, Shijun

• Atherosclerotic cardiovascular disease (CVD) is the leading cause of death worldwide. Long-term elevated low-density lipoprotein cholesterol (LDL-C) levels are the major risk factors for the development of atherosclerotic lesions, which can eventually lead to myocardial infarction and many strokes. Lowering LDL-C levels remains the cornerstone of CVD treatment. LDL receptor (LDLR) in the liver is mainly responsible for the removal of LDL-C from the blood. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by promoting LDLR degradation. An array of pharmaceutical companies has created therapeutic agents to inhibit PCSK9, including PCSK9 monoclonal antibodies and small interfering RNAs. These PCSK9 inhibitors, in combination with existing cholesterol-lowering drug statins, have been shown to significantly reduce circulating LDL-C levels and the risk of CVD in a wide variety of subjects. However, we still face considerable challenges in providing equitable access to this PCSK9 therapy. Studies of the cellular and molecular biology of PCSK9 can provide considerable insight into the development of novel PCSK9 inhibitors with efficacy and cost-effectiveness. This thesis aims to investigate the molecular biology of PCSK9, including the PCSK9 secretory pathway and, upon secretion PCSK9’s interaction with LDLR, as well as the molecular mechanism of PCSK9/LDLR lysosomal degradation.
  In chapter 3, I investigated the role of the C-terminal domain (CTD) of PCSK9 and SEC24, the cargo adaptor protein of the coated protein complex II (COPII), in PCSK9 maturation and secretion. I found that mutant PCSK91-528, in which amino acids from 528 to the end (amino acid 692) were deleted, was maturated and secreted from cells as efficiently as the wild-type protein. Conversely, mutants PCSK91-446, PCSK91-445, and PCSK91-444 that missing amino acids 447-692, 446-692, and 445-692, respectively, all significantly impaired PCSK9 maturation and secretion to a similar extent. Mutant PCSK91-444 essentially eliminated PCSK9 secretion. We also found that natural variants in the CTD, including S462P, S465L, E482G, R495Q and A522T, impaired PCSK9 secretion. Furthermore, knockdown of SEC24A, SEC24B, and SEC24C (not SEC24D) reduced the full-length PCSK9 secretion but had no effect on the secretion of mutant PCSK91-446. Together, these findings demonstrate the critical role of the CTD of PCSK9 and SEC24A, SEC24B, and SEC24C in PCSK9 maturation and secretion.
  In chapter 4, I identified the amino acid residues in the ligand-binding repeat domain (LRs) of LDLR important for PCSK9 binding at the cell surface. We have previously shown that, in addition to the epidermal growth factor precursor homology repeat-A (EGF-A) of LDLR, at least three ligand-binding repeats of LDLR are required for PCSK9-promoted LDLR degradation. However, how exactly the LRs contribute to PCSK9’s action on the receptor is not completely understood. Here, I found that substitution of Asp at position 172 in the linker between the LR4 and the LR5 of LDLR with Asn (D172N) reduced PCSK9 binding at pH 7.4 (mimic cell surface) but not at pH 6.0 (mimic endosomal environment). On the other hand, mutation of Asp at position 203 in the LR5 of LDLR to Asn (D203N) significantly reduced PCSK9 binding at both pH 7.4 and pH 6.0. These findings indicate that amino acid residues in the LRs of LDLR play an important role in PCSK9 binding to the receptor.
  In chapter 5, I used the yeast-two hybrid system to identify the interaction between PCSK9 CTD and prosaposin (PSAP), a secretory and lysosomal protein participating in glycosphingolipid degradation. In this study, I demonstrated that knockdown (KD) of PSAP in cultured hepatoma-derived cells led to significantly elevated cell surface LDLR abundance and LDL uptake. Further, knockdown of hepatic PSAP by AAV-shRNA increased LDLR levels in the liver and decreased plasma LDL-C levels in the wild-type mice but not the Ldlr-/- mice. Mechanistic studies showed that the effect of PSAP on LDLR was independent of PCSK9. On the other hand, data obtained from confocal microscopy and co-immunoprecipitation revealed a direct interaction between PSAP and LDLR. Further studies are ongoing to address the relevance and cellular mechanism by which PSAP induces LDLR degradation.
  In summary, the work performed during this Ph.D. thesis will shed light on the molecular mechanism of PCSK9-promoted LDLR degradation. Yet, continued research promises to provide further progress in the development of PCSK9 inhibitors.

• Subjects / Keywords

  • PCSK9
  • Cholesterol
  • LDLR

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-prby-0964

• License

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