Binding of human apoA-I[K107del] variant to TG-rich particles: implications for mechanisms underlying hypertriglyceridemia

Gorshkova, Irina N.; Mei, Xiaohu; Atkinson, David

doi:10.1194/jlr.m047241

Cited by 9 publications

(27 citation statements)

References 52 publications

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“…ANS binding increases its fluorescence emission due to decreased quenching by the solvent. Free WT apoA-I has low structural stability, high aggregating propensity and other molten-globular properties 28 and binds ANS, 31 causing increased emission (Fig. 2F).…”

Section: Resultsmentioning

confidence: 99%

“…28 Emission spectra of Trp and 1-anilino-8-naphthalenesulfonate (ANS) were recorded from free proteins using FluoroMax spectrofluorimeter following published protocols. 31 Hydrogen deuterium exchange mass spectrometry (HDX MS) was performed on free proteins essentially as described previously 50 using 55 μg/ml protein concentration at which apoA-I is fully monomeric. 29 Further details of the experiments, including Figures S1–S3 showing additional aspects of HDX MS data, are provided in the Supplementary Material.…”

Section: Methodsmentioning

confidence: 99%

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Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic?

Das

Wilson

Mei

et al. 2016

Journal of Molecular Biology

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ApoA-I, the major protein of plasma high-density lipoprotein, removes cellular cholesterol and protects against atherosclerosis. ApoA-I mutations can cause familial amyloidosis, a life-threatening disease wherein N-terminal protein fragments form fibrils in vital organs. To unveil the protein misfolding mechanism and to understand why some mutations cause amyloidosis while others do not, we analyzed the structure, stability and lipid-binding properties of naturally occurring mutants of full-length human apoA-I causing either amyloidosis (G26R, W50R, F71Y, L170P) or aberrant lipid metabolism (L159R). Global and local protein conformation and dynamics in solution were assessed by circular dichroism, fluorescence, and hydrogen-deuterium exchange mass spectrometry. All mutants showed increased deuteration in residues 14–22, supporting our hypothesis that decreased protection of this major amyloid “hot spot” can trigger protein misfolding. In addition, L159R showed local helical unfolding near the mutation site, consistent with cleavage of this mutant in plasma to generate the labile 1–159 fragment. Together, the results suggest that reduced protection of the major amyloid “hot spot”, combined with structural integrity of the native helix-bundle conformation, shift the balance from protein clearance to β-aggregation. A delicate balance between the overall structural integrity of a globular protein and the local destabilization of its amyloidogenic segments may be a fundamental determinant of this and other amyloid diseases. Furthermore, mutation-induced conformational changes observed in the helix bundle, which comprises N-terminal 75% of apoA-I, and its flexible C-terminal tail suggest the propagation of structural perturbations to distant sites via an unexpected template-induced ensemble-based mechanism, challenging the classical structure-based view.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic?

Das

Wilson

Mei

et al. 2016

Journal of Molecular Biology

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“…Mutation studies of lys107del and lys107A confirmed that the registry shift and ensuing disruption of the inter-helical salt bridges in apoA-I[Lys107del] result in destabilization of the helical bundle structure and greater exposure of hydrophobic surfaces. Opening of the N-terminal helical bundle in the apoA-I[Lys107del] variant facilitated its binding to TG-rich lipoproteins and thus, may reduce their lipolysis and contribute to the development of HTG in carriers of the mutation (54). …”

Section: Introductionmentioning

confidence: 99%

Lipid-free Apolipoprotein A-I Structure: Insights into HDL Formation and Atherosclerosis Development

Mei

Atkinson

2015

Archives of Medical Research

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Apolipoprotein A-I is the major protein in high-density lipoprotein (HDL) and plays an important role during the process of reverse cholesterol transport (RCT). Knowledge of the high-resolution structure of full-length apoA-I is vital for a molecular understanding of the function of HDL at the various steps of the RCT pathway. Due to the flexible nature of apoA-I and aggregation properties, the structure of full-length lipid-free apoA-I has evaded description for over three decades. Sequence analysis of apoA-I suggested that the amphipathic α-helix is the structural motif of exchangeable apolipoprotein, and NMR, X-ray and MD simulation studies have confirmed this. Different laboratories have used different methods to probe the secondary structure distribution and organization of both the lipid-free and lipid-bound apoA-I structure. Mutation analysis, synthetic peptide models, surface chemistry and crystal structures have converged on the lipid-free apoA-I domain structure and function: the N-terminal domain [1–184] forms a helix bundle while the C-terminal domain [185–243] mostly lacks defined structure and is responsible for initiating lipid-binding, aggregation and is also involved in cholesterol efflux. The first 43 residues of apoA-I are essential to stabilize the lipid-free structure. In addition, the crystal structure of C-terminally truncated apoA-I suggests a monomer-dimer conversation mechanism mediated through helix 5 reorganization and dimerization during the formation of HDL. Based on previous research, we have proposed a structural model for full-length monomeric apoA-I in solution and updated the HDL formation mechanism through three intermediate states. Mapping the known natural mutations on the full-length monomeric apoA-I model provides insight into atherosclerosis development through disruption of the N-terminal helix bundle or deletion of the C-terminal lipid-binding domain.

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“…While Rall et al found reduced efficiency to activate LCAT of isolated K107del in vitro [40], this function was shown similar to Wt in other studies [41] [42]. In a similar trend, lipid binding affinity was reported either decreased [41], or similar to Wt [43]. Cholesterol efflux from cultured cells was not significantly altered by the deletion [44] [45].…”

Section: Discussionmentioning

confidence: 56%

“…Cholesterol efflux from cultured cells was not significantly altered by the deletion [44] [45]. In other patients this mutant was associated withhypertriglyceridemia [39], and in vitro studies supported an increased binding to triglyceriderich particles [43]. These findings suggest that, in addition to the lower efficiency that the mutation may induce in the normal apoA-I functions, the effect does not seem to be drastic.…”

Section: Discussionmentioning

confidence: 78%

Understanding the role of apolipoproteinA-I in atherosclerosis. Post-translational modifications synergize dysfunction?

Ludovico¹,

Gisonno²,

González³

et al. 2020

Preprint

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Highlights-Oxidation is clue to induce protein misfolding -Natural mutation does not seem critical as a sole reason to determine pathogenicity -Atherosclerosis and amyloidosis are closely related -Intramolecular crosslinking restrains protein flexibility and function 4 Abstract Background: The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis. Methods:Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in the native conformation or under oxidation or intramolecular crosslinking.Results: K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants. Conclusions:We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding.Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency.General Significance: The results learnt from this variant strength a close association between amyloidosis and atherosclerosis.

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Binding of human apoA-I[K107del] variant to TG-rich particles: implications for mechanisms underlying hypertriglyceridemia

Cited by 9 publications

References 52 publications

Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic?

Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic?

Lipid-free Apolipoprotein A-I Structure: Insights into HDL Formation and Atherosclerosis Development

Understanding the role of apolipoproteinA-I in atherosclerosis. Post-translational modifications synergize dysfunction?

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