High‐efficient bacterial production of human ApoA‐I amyloidogenic variants

Journal of Lipid Research

Obici

et al. 2021

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Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) is essential for the transportation of cholesterol between peripheral tissues and the liver. However, specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis, the pathological accumulation of protein fibrils in tissues and organs. Carriers of these mutations do not exhibit increased cardiovascular disease risk despite displaying reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of patients carrying either L75P or L174S ApoA-I amyloidogenic variants a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.

Section: Methodsmentioning

confidence: 76%

“…Human ApoA-I proteins, containing a His-tag and tobacco etch virus (TEV) protease recognition site at the N terminus, were expressed in the bacterial Escherichia coli ) BL21(DE3) pLysS strain (Invitrogen, Thermo Fisher Scientific) as described in the study by Del Giudice and Lagerstedt ( 21 ).…”

Section: Methodsmentioning

confidence: 99%

Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux

Journal of Lipid Research

Obici

et al. 2021

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“…The recombinant proteins were isolated using an immobilized metal affinity chromatography (IMAC, His-Trap-Nickel-chelating columns, GE Healthcare, Chicago, IL, USA) and the his-tag removed by TEV protease treatment. The his-tag free proteins were then purified by performing a second IMAC [43]. Protein purity was analyzed by SDS-PAGE, followed by Coomassie staining, and protein concentration was determined by using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

The Apparent Organ-Specificity of Amyloidogenic ApoA-I Variants Is Linked to Tissue-Specific Extracellular Matrix Components

Giudice

et al. 2022

IJMS

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Apolipoprotein A-I (ApoA-I) amyloidosis is a rare protein misfolding disease where fibrils of the N-terminal domain of the protein accumulate in several organs, leading to their failure. Although ApoA-I amyloidosis is systemic, the different amyloidogenic variants show a preferential tissue accumulation that appears to correlate with the location of the mutation in the protein sequence and with the local extracellular microenvironment. However, the factors leading to cell/tissues damage, as well as the mechanisms behind the observed organ specificity are mostly unknown. Therefore, we investigated the impact of ApoA-I variants on cell physiology and the mechanisms driving the observed tissue specificity. We focused on four ApoA-I amyloidogenic variants and analyzed their cytotoxicity as well as their ability to alter redox homeostasis in cell lines from different tissues (liver, kidney, heart, skin). Moreover, variant-specific interactions with extracellular matrix (ECM) components were measured by synchrotron radiation circular dichroism and enzyme-linked immunosorbent assay. Data indicated that ApoA-I variants exerted a cytotoxic effect in a time and cell-type-specific manner that seems to be due to protein accumulation in lysosomes. Interestingly, the ApoA-I variants exhibited specific preferential binding to the ECM components, reflecting their tissue accumulation pattern in vivo. While the binding did not to appear to affect protein conformations in solution, extended incubation of the amyloidogenic variants in the presence of different ECM components resulted in different aggregation propensity and aggregation patterns.

“…Human ApoA-I proteins, containing a His-tag and tobacco etch virus (TEV) protease recognition site at the N-terminus, were expressed in the bacterial Escherichia coli ( E. coli ) BL21(DE3) pLysS strain (Invitrogen, Thermo Fisher Scientific) as described in 38 . Recombinant proteins were then purified using immobilized metal affinity chromatography (His-Trap-Nickel-chelating columns, GE Healthcare) followed by treatment with TEV protease and a second immobilized metal affinity chromatography step to remove the His-tag.…”

Section: Methodsmentioning

confidence: 99%

Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux

Lagerstedt

et al. 2020

Preprint

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24Specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are 25 responsible for a late-onset systemic amyloidosis. Carriers do not exhibit increased 26 cardiovascular disease risk despite reduced levels of ApoA-I/ HDL-cholesterol. To explain 27 this paradox, we show that the HDL particle profile of L75P and L174S patients presents a 28 higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as 29 well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to 30 catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and 31 hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered 32 secondary structure composition and display a more flexible binding to lipids compared to 33 their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I 34 amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles 35 and better cholesterol efflux due to altered, region-specific protein structure dynamics. 36 37 Keywords 38 Apolipoprotein A-I, amyloidosis, high-density lipoprotein, cholesterol efflux, cardiovascular 39 disease 40 41 Abbreviations 42 ABCA1, ATP binding cassette A1; ABCG1, ATP binding cassette G1, ApoA-I, apolipoprotein 43 A-I; ApoB, apolipoprotein B; CD, circular dichroism; CVD, cardiovascular disease; FC, 44 unesterified free cholesterol; HDL, high-density lipoprotein; HDX, hydrogen-deuterium 45 exchange; LCAT, lecithin-cholesteryl acyl transferase; PBS, phosphate buffer saline; POPC, 46 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; rHDL, reconstituted HDL; SRCD, 47 Synchrotron Radiation Circular Dichroism; TEV, tobacco etch virus; Tm, transition 48 temperature; WT, wild-type.49 3 Main 50 Amyloidoses are a broad group of diseases caused by protein instability and misfolding that 51 leads to pathological aggregation of proteins as amyloid deposits in tissues and organs 1 .52 These amyloid deposits are either localized, as in Alzheimer's and Parkinson's diseases, or 53 systemic, as is the case of transthyretin and light-chain amyloidoses. The different types of 54 amyloidoses commonly lead to severe and age-related damage of the tissues where 55 aggregation takes place. The understanding of the determinants leading to protein fibril 56 formation and amyloidosis development is thus key for finding ways to develop efficient 57 treatments for the affected individuals.58 Apolipoprotein A-I (ApoA-I), associated with hereditary systemic amyloidosis, has a key role 59 for the transportation of cholesterol and lipids in the circulation between peripheral tissues 60 and the liver 2 . Following its synthesis in the liver and intestines, ApoA-I mediates this 61 transportation by the formation of high-density lipoprotein (HDL) particles. These load 62 cholesterol and lipids from macrophages at the artery wall via a regulated efflux mechanism 63 catalyzed by the cellular ATP-binding cassette (ABC)A1 receptor. HDLs are then c...