A cycle of transport and regulation unveiling The hidden routes of cellular cholesterol transport

Cholesterol is an essential molecule that is required for, among other things, cellular membranes, bile acids, vitamin D, and steroid hormones. Conversely, excessive cholesterol levels can contribute to the development of various diseases, such as atherosclerosis. Therefore, cholesterol homeostasis requires tight regulation. Cholesterol can be acquired through dietary uptake or synthesized endogenously by cells. Certain cell types, such as hepatocytes, are also capable of secreting cholesterol in the form of very low-density lipoprotein (VLDL) particles, which are subsequently converted into low-density lipoprotein (LDL) particles in the circulation. The principal structural protein of both lipoprotein particles is apolipoprotein B (APOB). This thesis describes the cycle of cholesterol transport and the regulation of cholesterol secretion and uptake.
In this thesis, Chapter 1 provides a broad introduction to the cycle of VLDL formation and transport, the conversion of VLDL into LDL, the uptake of LDL via the LDL receptor (LDLR), and the subsequent intracellular trafficking of cholesterol from these particles to cellular organelles. Several aspects of cholesterol transport described in this chapter are investigated in more detail throughout this thesis.
Chapter 2 describes the development of a hepatic cell model in which APOB is endogenously tagged with the fluorescent protein mNeonGreen. We demonstrate that this tag does not interfere with the normal regulation of APOB and that this model is suitable for tracking intracellular APOB and quantifying its secretion. We further validate this model by identifying SYVN1 as the E3 ubiquitin ligase responsible for APOB degradation.
In Chapter 3, this cell model is used to perform a genetic screen aimed at identifying novel regulators of APOB. We identified several genes that had not previously been linked to APOB or VLDL metabolism. One of these genes, RIC1 (also known as KIAA1432), was selected for in-depth characterization. Cells lacking RIC1 have severely reduced VLDL secretion and APOB accumulation in lysosomes.
Chapter 4 introduces a novel cell model in which APOB is endogenously tagged with a miniTurbo proximity labeling enzyme. Similar to the approach used in Chapter 2, this small enzyme enables the biotinylation of proteins in close proximity to APOB. These biotinylated proteins can subsequently be isolated and identified. Using this approach, we identified numerous proteins located in the proximity of APOB. Although several known interactors were detected, the majority of the identified proteins have not previously been associated with APOB.
Chapter 5 presents a literature review on the post-transcriptional and post-translational regulation of the LDLR. Post-transcriptional regulation is discussed in terms of mRNA splicing, mRNA stability, and the role of microRNAs. For post-translational regulation, we describe multiple proteins that influence LDLR protein stability, including IDOL, GOLIATH, ASGR1, MT1-MMP, BMP1, and γ-secretase.
Chapter 6 describes an additional genetic screen aimed at identifying novel proteins involved in cholesterol uptake and the transport of cholesterol from lysosomes. One of the most notable findings was that Niemann–Pick disease type C1 (NPC1), a lysosomal protein previously thought to be essential for cholesterol transport, appeared to be unimportant in our cellular system. These cells therefore seem to utilize an alternative mechanism, which we aim to discover.
In Chapter 7, we further analyze the data obtained from the screen described in Chapter 6. Previously unidentified hits are validated, and the functions of several candidate proteins are investigated in greater detail.
Finally, Chapter 8 summarizes the results presented in the preceding chapters and provides a general discussion. Taken together, this thesis contributes to an improved understanding of intracellular cholesterol transport.