Hereditary ApoA-I-related amyloidosis: aggregation propensity in the presence of ECM components and modulation of cholesterol efflux

Abstract

Apolipoprotein A-I (ApoA-I) is the most predominant protein of high-density lipoprotein (HDL), which is an essential component for reverse cholesterol transport (RCT). ApoA-I has many health-promoting functions, such as anti-atherosclerosis and antioxidant properties; however, carriers of certain rare mutations in the APOA1 gene suffer from a disease called hereditary ApoA-I-related amyloidosis. Amyloidosis is the formation of β-strand aggregates of proteins or protein-fragments into fibrillar structures and plaques that may severely harm affected organs. Interestingly, AApoA-I variants have the tendency to aggregate preferentially in certain organs depending on the location of the mutation. Indeed, ApoA-I variants that carry mutations in the N-terminal domain tend to accumulate in liver and kidneys, whereas mutations outside this domain lead to the accumulation of protein fibrils in heart and skin.

To test the hypothesis that the variants’ tissue-specificity is due to differing interactions with the organ-specific extracellular environments, we used Synchrotron Radiation Circular Dichroism (SRCD) and Thioflavin-T (ThT) assays to elucidate if the extracellular matrix (ECM) components heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronate were able to induce specific conformational change in four ApoA-I amyloidogenic variants. G26R and L75P were selected to represent amyloid formation in the liver and kidneys, and L174S and L178H were selected to represent heart and skin. We found that tissue-specific ECM components do tend to cause conformational change and induce more formation of α-amyloid in protein variants with a tendency to accumulate in those tissues. The ECM components did not cause significant change in the secondary structure of the ApoA-I variants at short-term incubation, but over a longer period the formation of β-strands, and thereby, aggregation propensity, was changed.

Moreover, recent studies have shown that carriers of ApoA-I amyloidogenic mutations have lower levels of HDL, yet do not show increased risk of cardiovascular diseases (CVD). Cholesterol efflux experiments were therefore carried out to determine if G26R, L75P, L174S, or L178H amyloidogenic variants mediate efflux of cholesterol from J774 macrophages more efficiently than wild-type ApoA-I. The variants of ApoA-I were found to have a higher affinity to cholesterol despite a lower cholesterol binding capacity, therefore suggesting that the changes in amyloidogenic ApoA-I protein allow for more efficient removal of cholesterol from cells. Further investigation is needed to solidify the statistical results of these data.

Popular Abstract

ApoA-I-related amyloidosis: a disease with both a good and a bad side

Hereditary apolipoprotein A-I related amyloidosis is a rare disease that begins affecting patients later in life. It is caused by accumulation of ApoA-I proteins in certain organs due to mutations passed on through family members and can eventually cause organ failure. We would like to find out more about the interactions that lead to this disease, as well as understand a potentially positive side-effect of these mutations.

Apolipoprotein A-I (ApoA-I) is the protein that makes up the major part of what is known as the “good” cholesterol, high-density lipoprotein (HDL). ApoA-I in the form of HDL is part of the process in which cholesterol is removed from tissues in the body and brought to the liver for disposal. ApoA-I proteins travel around the body in the bloodstream until they come upon cells with a receptor, think of it as ApoA-I as a key and the receptor as a lock, that when bound releases fats and cholesterols to the protein. In conjunction with other ApoA-I molecules, an HDL “sphere” is formed, filled with fats and cholesterols. When the HDL sphere is full it travels to the liver, where it releases the cholesterol to be recycled or disposed of. By removing unwanted cholesterol, especially from the arteries of the heart, this mechanism provides protective properties against cardiovascular disease.

There is, however, a bad side to this protein along with its protective properties. A rare, hereditary disease called ApoA-I-related amyloidosis is found in people who have certain mutations, or variations, in the gene that encodes this protein. Amyloidosis is a cause of many well-known diseases, such as Alzheimer’s or Parkinson’s disease, and means that protein collects into plaques that can sometimes disturb the functions of organs. These proteins have changed shape,
causing them to stick to each other and form into strong strands of what are called amyloid fibrils in the spaces between cells. When many amyloid fibrils join, they are called amyloid plaques. Unlike Alzheimer’s or Parkinson’s, ApoA-I-related amyloidosis does not generally affect the brain or nerve function, but instead accumulate in certain organs depending on the location of the mutation in the protein. Changes closer to the “beginning” of the protein usually result in accumulation of protein in the liver and kidneys. Changes near the “end” of the protein tend to result in accumulation of amyloid in the heart and skin. Depending on the organ afflicted by amyloid plaques, this disease can either cause mild symptoms or result in organ-failure later in life. However, another interesting, less harmful, characteristic of carriers of one of these amyloidogenic variants is that they tend to have lower levels of HDL in their blood. In people without this disease, low HDL is usually a risk-factor for a higher chance of developing cardiovascular disease. However, although these patients have less HDL they do not tend to have more cardiovascular disease, and the reason for this is still unclear.

This thesis aims to find out if extracellular matrix (ECM) components, which are types of sugars that exist in the spaces between cells, interact with ApoA-I variants in a way that results in increased aggregation. It also aims to discover if the amyloidogenic ApoA-I proteins are better at removing cholesterol from cells in the arteries and therefore mitigate the risk of cardiovascular disease despite having lower levels of HDL.

ECM Components

Previously, scientists in the lab I did my thesis work in determined that the proteins that tend to accumulate mainly in the liver, kidneys, and sometimes reproductive organs, and G26R and L75P, and the proteins that tend to accumulate in heart and skin, L174S and L178H, bind to ECM components that correspond to the tissues they are derived from.

By measuring how a type of circular light is absorbed we can figure out if the conformation, or shape, of the proteins is changed in the presence of ECM components in comparison to the regular protein. We can also use a fluorescent molecule to measure this change. The conformation of this protein will change when it begins to form amyloid fibrils. Overall, we discovered that the proteins that gather in the heart and skin tend to change conformation and stick together when incubated together with ECM components from those tissues. These components can also occur in the reproductive organs, and, interestingly, we saw that L75P also began to change when incubated together with these.

Cholesterol efflux

To find out why patients with ApoA-I-related amyloidosis have less HDL but not more heart disease, we used a type of experiment in which we load cells with equal amounts of cholesterol and then, after incubating them with our protein, measure how quickly and how much cholesterol each protein has removed. Our results suggest that amyloidogenic ApoA-I can remove cholesterol more quickly than the normal protein, but we must do more experiments to solidify these results.


Master’s Degree Project in Molecular Biology 60 credits 2019
Department of Medicine, Lund University
Advisor: Jens Lagerstedt & Rita Del Giudice
Medical Protein Science, Experimentell Medicinsk Vetenskap

• author: Lindvall, Mikaela
• supervisor: Jens Lagerstedt; Rita Del Giudice
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20182
• year: 2019
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8990029
• date added to LUP: 2019-07-04 14:11:49
• date last changed: 2019-07-04 14:11:49