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Insights into Membrane Type-1 Matrix Metalloproteinase cleavage of the Low-density Lipoprotein Receptor
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- Author / Creator
- Alabi, Adekunle
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The Low-density lipoprotein receptor (LDLR) mediated cellular uptake of LDL is the main pathway for plasma LDL cholesterol (LDL-C) clearance. However, the LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into the extracellular milieu and as such reducing its LDL-C clearance efficiency. Plasma sLDLR levels are positively correlated with plasma LDL-C levels. Membrane type 1-matrix metalloproteinase (MT1-MMP) is a Zn2+-dependent endopeptidase that can cleave extracellular matrix and non-matrix substrates.
The work presented in this thesis investigates, the proteinase responsible for LDLR cleavage and as such identifies MT1-MMP as a key metalloproteinase responsible for LDLR shedding. We found that knockdown of MT1-MMP increased cellular LDLR abundance and reduced the levels of sLDLR in cultured hepatocytes. Interaction between both proteins was ascertained by their co-immunoprecipitation and co-localization in hepatoma cell lines. Consistently, mice lacking hepatic MT1-MMP displayed an increase in cellular LDLR levels and a corresponding reduction in plasma levels of sLDLR, HDL-cholesterol, and non-HDL cholesterol. Opposite effects were observed when MT1-MMP was overexpressed. We also demonstrated that hepatocyte-specific overexpression of MT1-MMP significantly increased atherosclerotic lesion area in Apoe knockout mice.
In addition, we found that the majority of circulating sLDLR were associated with apoB and apoE-containing lipoproteins in both mouse and human plasma. The sLDLR retains its ability to bind to LDLR ligands and as such reduces ligands available to be cleared by the LDLR. Combined treatment of MT1-MMP knockdown and inhibition of other LDLR regulating pathways such as statin inhibition of HMG-CoA reductase, inhibition of PCSK9 and γ-secretase, had a combined beneficial increase on LDLR levels invitro accompanied with an improved clearance of circulating cholesterol in vivo with statin treatment.
It was determined that MT1-MMP cleaves the LDLR possibly at multiple sites within the protein. Deletion of various regions on the protein did not abrogate its MT1-MMP cleavage nor did mutation of cleavage sites as determined by CleavePredict affect LDLR cleavage by MT1-MMP. MT1-MMP cleaved LDLR structurally related proteins such as ApoER2 and VLDLR. Similarly, MT1-MMP structurally related metalloproteinase MT2-MMP cleaves the LDLR but with a lesser efficiency as compared to MT1-MMP.
We identified a variant of MT1-MMP (rs139288377) in the Dallas Heart Study with the mutation A37P; this variant was significantly associated with plasma LDL-C levels. The average LDL-C levels of 36 people with the variant were 87 mg/dl, compared with 110 mg/dl in the control group. Our experiments showed that this mutation reduces the ability of MT1-MMP to efficiently cleave the LDLR.
Thus, this thesis demonstrates that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis. -
- Graduation date
- Spring 2021
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.