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Cited by 24Protein Structure and DynamicsLipid Membrane Structure and BehaviorLipoproteins and Cardiovascular Health

Biophysical Analysis of Apolipoprotein E3 Variants Linked with Development of Type III Hyperlipoproteinemia

Open access
Georgiadou, Dimitra, Chroni, Angeliki, Vezeridis, Alexander, Zannis, Vassilis I., Stratikos, Efstratios·2011·PLoS ONE
DOI 10.1371/journal.pone.0027037PMC PMC3206067
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Abstract

BACKGROUND: Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent. CONCLUSIONS/SIGNIFICANCE: Overall, our findings suggest that single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.

AI snapshot

Written from the paper’s full text.

TL;DR

This study biophysically characterized three type III HLP–associated apoE3 variants (R136S, R145C, K146E) and found small, variant-specific changes in stability, oligomerization, hydrophobic exposure, and lipid-remodeling kinetics. The changes were non-concerted, making a single unifying biophysical mechanism for type III HLP pathogenesis unlikely.

Key findings

  • Circular dichroism showed no significant secondary-structure changes: α-helical content at 222 nm was 58.7±3.2% for WT, 56.8±1.2% for R145C, 59.7±2.2% for K146E, and 59.4±4.9% for R136S.
  • Thermal unfolding revealed variant-specific effects: K146E had a higher Tm (55.7±0.1°C vs WT 53.6±0.2°C, p=0.002), R136S had a lower Tm (51.9±0.4°C, p=0.004), and R145C had unchanged Tm but reduced cooperativity (n=6.9±0.4 vs WT 7.7±0.2) and apparent ΔH (21.3±1.4 vs WT 27.9±0.1 kcal/mol). R136S and K146E also showed 95–99% signal recovery upon thermal refolding, whereas WT recovered about 60%.
  • Chemical denaturation showed K146E had higher total apparent ΔG (6.0±0.3 vs WT 4.4±0.2 kcal/mol, p=0.08), while R145C and R136S did not differ significantly from WT. ANS binding was increased only for R145C (4.2±0.4-fold vs WT 3.6±0.5, p=0.03).
  • DLS indicated R136S had a larger hydrodynamic diameter (16.9±2.1 nm vs WT 10.4±1.7 nm), and DMPC remodeling showed slower slow-phase half-lives for R145C (1416±69 sec, p=0.012) and R136S (1378±178 sec, p=0.004) compared with WT (1039±143 sec).

Methods

  • Recombinant wild-type apoE3 and variants R136S, R145C, and K146E were expressed in HTB13 cells via adenovirus, purified by a lipidation/delipidation protocol, and refolded before analysis.
  • Biophysical assays included far-UV circular dichroism for secondary structure and thermal denaturation, Trp fluorescence for guanidine hydrochloride chemical denaturation, ANS fluorescence for hydrophobic surface exposure, DMPC vesicle remodeling kinetics by absorbance at 325 nm, and dynamic light scattering for solution oligomerization.
  • Data were fitted with Graphpad Prism; paired Student’s t-tests compared separately refolded protein batches, with 3–4 experiments per measurement.

Limitations

  • The paper states that apoE3 thermal unfolding is not fully reversible due to aggregation, and thermal parameters were calculated assuming a reversible two-state model.
  • The authors state that without knowing the protein’s folding/unfolding pathways, the observed phenomena cannot be unequivocally explained; the small magnitude and non-concerted nature of the changes make it unlikely that they constitute a primary unifying mechanism for type III HLP.
  • Evident from the text: this is an in vitro biophysical study of lipid-free recombinant apoE3 variants, with no direct LDL-receptor/HSPG binding assays or in vivo disease outcomes, and the conclusions rely on 3–4 independent experiments per assay.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0027037

Cites

76 of 76 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

17 of 58 cited sources checked · 5 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    The polymorphism at position 112 brings about alterations in the thermodynamic properties of the molecule that may underlie functional changes linked with disease predisposition.

    “This polymorphic variation brings about alterations in the thermodynamic properties of the molecule that may underlie functional changes linked with disease predisposition [61], [65], [71], [72], [73], [74].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [61] CY Chou, 2006, Structural and functional variations in human apolipoprotein E3 and E4., J Biol Chem source

      The paper identifies the position-112 Cys-to-Arg difference between apoE3 and apoE4 and reports isoform-specific structural and functional differences. Its concluding passage links apoE4’s potential structural stability to pathophysiologic functions, but our copy does not establish that the position-112 substitution alters thermodynamic properties specifically. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [65] JA Morrow, 2000, Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain., Biochemistry source
    • cites [71] JA Morrow, 2002, Apolipoprotein E4 forms a molten globule. A potential basis for its association with disease., J Biol Chem source
  • Premisecitation

    Heterozygosity for apoE3[K146E] with the apoE3 allele results in dominant expression of type III HLP.

    “Finally, heterozygosity for the apoE variant apoE3[K146E] with the apoE3 allele resulted in a dominant expression of type III HLP [33], [34], [35].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [33] WA Mann, 1989, Apolipoprotein E-1Harrisburg: a new variant of apolipoprotein E dominantly associated with type III hyperlipoproteinemia., Biochim Biophys Acta source
    • cites [34] K Moriyama, 1992, Apolipoprotein E1 Lys-146----Glu with type III hyperlipoproteinemia., Biochim Biophys Acta source
    • cites [35] WA Mann, 1995, Dominant expression of type III hyperlipoproteinemia. Pathophysiological insights derived from the structural and kinetic characteristics of ApoE-1 (Lys146–>Glu)., J Clin Invest source
  • Premisecitation

    In the newly published structure of a monomeric mutant of apoE3 (2L7B), the N-terminal segment is folded in a four-helix bundle with the locations of R136, R145 and K146 in similar configurations as in structure 1LPE.

    “In this structure (pdb code: 2L7B) the N-terminal segment of the protein is also folded in a four-helix bundle (Figure 9A) with the locations of amino acids R136, R145 and K146 in similar configurations as in structure 1LPE.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [68] J Chen, 2011, Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions., Proc Natl Acad Sci U S A source

      The Results describe the monomeric apoE3 mutant and its N-terminal four-helix bundle, and the data-deposition footnote identifies the structure as PDB 2L7B. The Results say the bundle is broadly similar to isolated N-terminal structures, but our copy does not establish that R136, R145, and K146 have similar configurations to those in 1LPE. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation

    Heterozygosity for apoE3[R136S] with the apoE2 allele results in type III HLP.

    “Specifically, heterozygosity for the apoE variant apoE3[R136S] (previously designated apoE2 Christchurch) with the apoE2 allele results in type III HLP [27].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] MR Wardell, 1987, Apolipoprotein E2-Christchurch (136 Arg----Ser). New variant of human apolipoprotein E in a patient with type III hyperlipoproteinemia., J Clin Invest source

      The Introduction and Discussion report a patient with type III HLP who carried both apoE2 (158 Arg→Cys) and apoE2-Christchurch (136 Arg→Ser), supporting the reported association. But the Introduction says an apoE2 variant alone is not sufficient to cause type III HLP and proposes an additional genetic defect. The source therefore does not establish that this allele combination necessarily results in the disease. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    In the 2L7B structure, the C-terminal domain is highly helical and folded around the N-terminal domain so as to completely protect the LDL-receptor binding region 136-150 from the solvent.

    “The C-terminal domain is highly helical and folded around the N-terminal domain in such a configuration to completely protect the LDL-receptor binding region 136-150 from the solvent (Figure 9B).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [68] J Chen, 2011, Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions., Proc Natl Acad Sci U S A source

      The Results (page not given) describe the C-terminal domain’s helices interacting with the N-terminal domain and shielding the major LDLR-binding region. But Table 2 reports small, nonzero accessible surface areas for several residues from 136 to 150, so the claim that the region is completely protected from solvent overstates the evidence. The paper identifies 2L7B as its deposited structure. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 9
  • May combine two claims. 20
  • The quote only partly states this claim. 6

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The N-terminal domain of apoE is folded into a four-helix bundle of amphipathic alpha-helices spanning residues 24-164, stabilized by hydrophobic interactions and salt bridges.

    “X-ray crystallography studies showed that the N-terminal domain is folded into a four-helix bundle of amphipathic α-helices spanning residues 24-164 that are stabilized by hydrophobic interactions and salt bridges [15], [16], [17].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [15] C Wilson, 1991, Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E., Science source
    • cites [16] LM Dong, 1994, Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms., J Biol Chem source

      The Abstract and the structural description (p. 22358) report an X-ray-derived four-helix bundle in the amino-terminal fragment; the structural description also says residues 1–22 and 165–191 were disordered. Our copy does not specify a 24–164 span or describe the bundle as amphipathic and stabilized by hydrophobic interactions. It identifies a particular salt bridge in apoE4, not salt bridges as a general stabilizing feature of the bundle. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [17] C Wilson, 1994, Salt bridge relay triggers defective LDL receptor binding by a mutant apolipoprotein., Structure source

  • Classified as probably not a substantial claim.

    Premisecitation

    Inter-domain interactions within apoE have been hypothesized to underlie key conformational changes related to apoE function.

    “Inter-domain interactions within apoE have been hypothesized to underlie key conformational changes related to apoE function [44], [45].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [44] DM Hatters, 2006, Apolipoprotein E structure: insights into function., Trends Biochem Sci source
    • cites [45] RW Mahley, 2009, Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS., J Lipid Res source

  • May combine two claims.

    Interpretationcitation

    R145C is expected to destabilize the interaction between amphipathic helices N1 and 4, possibly leading to partial exposure of the intra-helix core.

    “Furthermore, variant R145C was found to expose additional hydrophobic surfaces to the solvent and it is expected to destabilize the interaction between amphipathic helices N1 and 4 possibly leading to partial exposure of the intra-helix core[68].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [68] J Chen, 2011, Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions., Proc Natl Acad Sci U S A source

      The Results describe exposed hydrophobic residues in apoE3’s CT domain, and Table 1 lists contacts involving residues near 145, but neither passage addresses R145C. Searches for R145C and for the proposed N1–Helix 4 destabilization and core exposure found no passage supporting those variant-specific effects. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Functional differences between apoE4 and apoE3 have been attributed to altered inter-domain interactions brought about by the polymorphism at position 112 of the N-terminal domain.

    “Specifically, functional differences between apoE4 and apoE3 have been attributed to altered inter-domain interactions brought about by the polymorphism at position 112 of the N-terminal domain.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [61] CY Chou, 2006, Structural and functional variations in human apolipoprotein E3 and E4., J Biol Chem source

      The introduction identifies the Cys112-to-Arg112 difference between apoE3 and apoE4, and the Discussion links their functional differences to a possible domain–domain interaction (page unavailable in our copy). But the paper says this interaction may assist apoE4 stability and that the proposed Arg61–Glu255 interaction cannot explain its observations; it does not establish that the position-112 change causes the functional differences through altered inter-domain interactions. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [65] JA Morrow, 2000, Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain., Biochemistry source
    • cites [71] JA Morrow, 2002, Apolipoprotein E4 forms a molten globule. A potential basis for its association with disease., J Biol Chem source

  • Classified as probably not a substantial claim.

    Premisecitation

    Structural and biophysical properties of apoE can dictate the function of the protein.

    “Several studies have suggested that the structural and biophysical properties of apoE can dictate the function of the protein and have provided insight into the mechanisms by which apoE is involved in cardiovascular and neurological diseases [42], [43], [44], [45], [46], [47], [48], [49].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [42] M Tanaka, 2006, Effect of carboxyl-terminal truncation on structure and lipid interaction of human apolipoprotein E4., Biochemistry source
    • cites [43] A Chroni, 2008, Biophysical analysis of progressive C-terminal truncations of human apolipoprotein E4: insights into secondary structure and unfolding properties., Biochemistry source
    • cites [44] DM Hatters, 2006, Apolipoprotein E structure: insights into function., Trends Biochem Sci source

  • May combine two claims.

    Premisecitation

    Homozygosity for apoE3[R136S] and heterozygosity for apoE3[R136S] with all apoE alleles also result in type III HLP, but disease penetrance is incomplete.

    “Additionally, it has been shown that homozygosity for apoE3[R136S] and heterozygocity for apoE3[R136S] with all apoE alleles result also in type III HLP, but the penetrance of the disease is incomplete [29].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] M Pocovi, 1996, Incomplete dominance of type III hyperlipoproteinemia is associated with the rare apolipoprotein E2 (Arg136->Ser) variant in multigenerational pedigree studies., Atherosclerosis source

      The Abstract reports one homozygote and four heterozygotes with the Arg136→Ser variant, and says linkage analysis suggested an association with incomplete dominance of type III hyperlipoproteinemia. It does not establish that heterozygosity with all other apoE alleles results in type III HLP; the citing sentence also states the outcome more definitively than the source does. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that the variants may carry additional properties contributing to pathogenic potential.

    “However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [28] A Lalazar, 1988, Site-specific mutagenesis of human apolipoprotein E. Receptor binding activity of variants with single amino acid substitutions., J Biol Chem source
    • cites [37] SC Rall Jr, 1992, The role of apolipoprotein E genetic variants in lipoprotein disorders., J Intern Med source
    • cites [38] DA Chappell, 1989, High receptor binding affinity of lipoproteins in atypical dysbetalipoproteinemia (type III hyperlipoproteinemia)., J Clin Invest source

  • May combine two claims.

    Findingown data

    Binding of ANS to apoE3 results in a marked increase of its fluorescence signal accompanied by a blue-shift, consistent with ANS binding to hydrophobic sites on the protein.

    “Binding of ANS to apoE3 resulted in a marked increase of its fluorescence signal accompanied by a blue-shift consistent with ANS binding to hydrophobic sites on the protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The R145C variant has a similar hydrodynamic diameter to wild-type apoE3 but with a slightly larger distribution.

    “The R145C variant had a similar hydrodynamic diameter but with a slightly larger distribution possibly indicating a tendency towards aggregation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ApoE3-K146E had a chemical denaturation free energy change of 6.0±0.3 kcal/mol, about 1.6 kcal/mol higher than wild-type apoE3 (p=0.08), indicating stabilization of this variant compared to wild-type.

    “ApoE3-K146E however had a ΔG of 6.0±0.3 kcal/mol, a value that was about 1.6 kcal/mol higher than the wild-type protein (p = 0.08) indicating stabilization of this variant compared to the wild-type protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ApoE3-R145C and apoE3-R136S had chemical denaturation free energy changes of 4.6±0.2 and 4.8±0.2 kcal/mol, respectively, values that did not statistically differ from wild-type apoE3 (4.4±0.2 kcal/mol).

    “ApoE3-R145C and apoE3-R136S had a ΔG of 4.6±0.2 and 4.8±0.2 kcal/mol respectively, values that did not statistically differ from the value calculated for the wild-type protein (4.4±0.2 kcal/mol).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In all DLS measurements, more than 99% of the scattering mass originated from species in the 10-20 nm diameter range, with less than 1% of the mass corresponding to higher-order aggregates.

    “In all cases >99% of the scattering mass was found to originate from species in the 10–20 nm diameter range, with <1% of the mass corresponding to higher-order aggregates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The second unfolding transition of wild-type apoE3 demonstrated similar sigmoidal shape and Tm to the first, indicating that part of the protein ensemble was able to undergo a reversible transition.

    “The second unfolding transition however, did demonstrate similar sigmoidal shape and Tm to the first indicating that part of the protein ensemble was able to undergo a reversible transition.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    R145C has a higher slow component half-life (1416±69 sec) than wild-type apoE3 (1039±143 sec) in DMPC remodeling, with p=0.012.

    “R145C | 133±41 | 1416±69 * | 24±3”
    • Also stated in: § DMPC remodeling kinetics · plain
      “*p = 0.012, **p = 0.004.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    R145C has increased ANS binding (4.2±0.4 fold) compared to wild-type apoE3 (3.6±0.5 fold), with p=0.03.

    “R145C | 0.9±0.2 | 3.6±0.2 | 4.6±0.2 | 2.5±0.2 | 1.6±0.2 | 0.36±0.08 | 2.33±0.04 *4 | 4.2±0.4 *6”
    • Also stated in: § Chemical denaturation measurements · plain
      “*1p = 0.07, *2p = 0.08, *3p = 0.08, *4p = 0.024, *5p = 0.06, *6p = 0.03”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    R136S has a higher slow component half-life (1378±178 sec) than wild-type apoE3 (1039±143 sec) in DMPC remodeling, with p=0.004.

    “R136S | 165±85 | 1378±178** | 15±4”
    • Also stated in: § DMPC remodeling kinetics · plain
      “*p = 0.012, **p = 0.004.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    R145C has a higher mid-transition point xiu (2.33±0.04 M) than wild-type apoE3 (2.09±0.03 M), with p=0.024.

    “R145C | 0.9±0.2 | 3.6±0.2 | 4.6±0.2 | 2.5±0.2 | 1.6±0.2 | 0.36±0.08 | 2.33±0.04 *4 | 4.2±0.4 *6”
    • Also stated in: § Chemical denaturation measurements · plain
      “*1p = 0.07, *2p = 0.08, *3p = 0.08, *4p = 0.024, *5p = 0.06, *6p = 0.03”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    K146E has a higher apparent ΔGtotal (6.0±0.3 kcal/mol) than wild-type apoE3 (4.4±0.2 kcal/mol), with p=0.08.

    “K146E | 1.0±0.2 | 4.9±0.3 *1 | 6.0±0.3 *2 | 2.7±0.2 | 2.1±0.3 *3 | 0.39±0.03 | 2.32±0.03 *5 | 3.9±0.4”
    • Also stated in: § Chemical denaturation measurements · plain
      “*1p = 0.07, *2p = 0.08, *3p = 0.08, *4p = 0.024, *5p = 0.06, *6p = 0.03”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    K146E has a higher apparent ΔG2 (4.9±0.3 kcal/mol) than wild-type apoE3 (3.4±0.1 kcal/mol), with p=0.07.

    “K146E | 1.0±0.2 | 4.9±0.3 *1 | 6.0±0.3 *2 | 2.7±0.2 | 2.1±0.3 *3 | 0.39±0.03 | 2.32±0.03 *5 | 3.9±0.4”
    • Also stated in: § Chemical denaturation measurements · plain
      “*1p = 0.07, *2p = 0.08, *3p = 0.08, *4p = 0.024, *5p = 0.06, *6p = 0.03”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For thermal and chemical denaturation measurements as well as ANS binding, statistical analysis was performed using experimental data collected on separate days.

    “For thermal and chemical denaturation measurements as well as ANS binding, statistical analysis was performed using experimental data collected on separate days.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    For DMPC remodeling measurements, all experiments were performed on the same day to minimize variability from the preparation of the DMPC vesicles.

    “For DMPC remodeling measurements all experiments were performed on the same day to minimize variability from the preparation of the DMPC vesicles.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    To test reproducibility, a paired Student t-test was applied to experimental data collected from separately refolded protein batches.

    “To test the reproducibility of the results we applied a paired student t-test to experimental data collected from separately refolded protein batches.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The predominantly reversible thermal transitions of R136S and K146E suggest that these variants may have different competing kinetics between refolding and thermally induced aggregation, indicative of changes in conformational plasticity.

    “This finding suggests that these variants may have different competing kinetics between refolding and thermally induced aggregation, indicative of changes in the conformational plasticity of the protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Differences in function and disease predisposition are established between apoE3 and apoE2, although these two variants differ only at a single amino acid at position 158.

    “Similarly, differences in function and disease predisposition are established between apoE3 and apoE2 although these two variants differ only at a single amino acid at position 158 [65], [72], [74].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [65] JA Morrow, 2000, Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain., Biochemistry source
    • cites [72] P Acharya, 2002, Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism., Biochim Biophys Acta source
    • cites [74] Y Huang, 2010, Mechanisms linking apolipoprotein E isoforms with cardiovascular and neurological diseases., Curr Opin Lipidol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Successful mutagenesis was confirmed by sequencing the template vector.

    “Successful mutagenesis was confirmed by sequencing the template vector.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Homozygosity and heterozygosity for apoE3[R145C] are associated with severe type III HLP.

    “Homozygosity and heterozygocity for the apoE variant apoE3[R145C] has also been associated with severe type III HLP [30], [31].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [30] SC Rall Jr, 1982, Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects., Proc Natl Acad Sci U S A source
    • cites [31] WJ de Villiers, 1997, The apolipoprotein E2 (Arg145Cys) mutation causes autosomal dominant type III hyperlipoproteinemia with incomplete penetrance., Arterioscler Thromb Vasc Biol source

  • May combine two claims.

    Interpretationown data

    Single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences.

    “Overall, our findings suggest that single amino acid changes in the functionally important region 136–150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences.”
    • Also stated in: § Introduction · hedged
      “Overall, our data point to changes in some of the biophysical parameters of each variant compared to the wild-type form suggesting that even single-amino acid changes in this region of the protein can influence the stability and conformation of apoE3.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    The R145C, K146E and R136S mutations are all located within helix 4 of the N-terminal four-helix bundle and encode non-conservative amino acid substitutions.

    “The R145C, K146E and R136S mutations are all located within helix 4 of the N-terminal four-helix bundle of the protein (Figure 1) and encode for non-conservative amino acid substitutions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Mutations in the 136-150 region of the N-terminal domain of apoE reduce LDL receptor binding capacity and have been linked with lipoprotein disorders such as type III hyperlipoproteinemia in humans.

    “Mutations in the 136–150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [22] R Mahley, 2001, Type III hyperlipoproteinemia (dysbetalipoproteinemia): The role of apolipoprotein E in normal and abnormal lipoprotein metabolism;

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [26] VIKD Zannis, 1993, Genetic mutations affecting human lipoproteins, their receptors, and their enzymes., Adv Hum Genet source

  • May combine two claims.

    Interpretationreasoning

    These inter-domain interactions are expected to be abrogated in the variants under study, perturbing important domain interactions in apoE3.

    “These interactions are expected to be abrogated in the variants under study here, essentially perturbing important domain interactions in apoE3.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In the N-terminal four-helix bundle structure of apoE3 (1LPE), the amino acid positions studied here (R136, R145, K146) are all largely exposed to the solvent and are thought to mediate interactions with the LDL receptor.

    “In this structure, the amino acid positions studied here are all largely exposed to the solvent and are thought to mediate interactions with the LDL receptor.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites 1LPE

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Each experimental set comprised both the wild-type protein and all three variants.

    “Each experimental set was comprised by both the wild-type (WT) protein and all three variants.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    SDS-PAGE analysis indicated that all four recombinant proteins migrated with identical profiles and were more than 98% pure.

    “SDS-PAGE analysis indicated that all four recombinant proteins migrated with identical profiles and were more than 98% pure (Figure 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    These apoE3 variants maintain their ability to predispose to type III HLP and do so in a dominant fashion, unlike apoE2.

    “Still, these variants not only maintain their ability to predispose to type III HLP, but also do so in a dominant fashion, unlike apoE2 [22], [26], [30], [31], [33], [34], [35].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [22] R Mahley, 2001, Type III hyperlipoproteinemia (dysbetalipoproteinemia): The role of apolipoprotein E in normal and abnormal lipoprotein metabolism;

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [26] VIKD Zannis, 1993, Genetic mutations affecting human lipoproteins, their receptors, and their enzymes., Adv Hum Genet source
    • cites [30] SC Rall Jr, 1982, Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects., Proc Natl Acad Sci U S A source

  • May combine two claims.

    Premisereasoning

    In many cases, the studied molecular properties of apoE variants associated with disease do not correlate well with disease penetrance or severity, indicating that other factors may also contribute to the pathogenesis of the disease.

    “In many cases the studied molecular properties of apoE variants associated with the disease do not correlate well with disease penetrance or severity indicating that other factors may also contribute to the pathogenesis of the disease.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [22] R Mahley, 2001, Type III hyperlipoproteinemia (dysbetalipoproteinemia): The role of apolipoprotein E in normal and abnormal lipoprotein metabolism;

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [75] KH Weisgraber, 1982, Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine-arginine interchange at a single site., J Biol Chem source
35 of 35

  • The authors state this as an assumption.

    Thermal unfolding parameters were calculated assuming a reversible two-state model.

    “1parameters calculated assuming a reversible two-state model.”

  • The paper relies on this without support.

    The in vitro biophysical alterations observed for the apoE3 variants are relevant to their pathogenic potential in type III HLP.

    “In this study we hypothesized that type III HLP predisposing mutations may lead to alterations in the structure and thermodynamic stability of apoE3 since such alterations have previously been linked with the pathogenic potential of the protein.”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • ApoE3 variants R136S, R145C, and K146E, associated with type III hyperlipoproteinemia but not with LPG, display much milder repercussions on apoE3 structure and stability.

    “ApoE3 variants R136S, R145C, and K146E, which have been associated with type III hyperlipoproteinemia, but not with LPG, display much milder repercussions on apoE3 structure and stability (34)”

    From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [34]

    The Introduction links R136S, R145C, and K146E to type III hyperlipoproteinemia, and the Discussion describes the magnitude of their structural and stability perturbations as relatively small. Our copy does not establish that these variants are unassociated with LPG or compare their effects with those of LPG-associated variants (searched: LPG, lipoprotein glomerulopathy). (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Thermal denaturation analysis was used as a method of comparing changes between apoE3 variants despite the process being gradual multistate and not fully reversible, because irreversible changes are relatively slow compared with heat unfolding.

    “Although the thermal denaturation of apoE3 is a gradual multistate and not fully reversible process, such analysis has been used before as a method of comparing changes between apoE3 variants, primarily because the irreversible changes are relatively slow compared with the heat unfolding (34,35).”

    From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [34]

    Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

  • The observed structural and thermodynamic alterations for all LPG-associated apoE3 mutants described here and in Georgiadou et al. (18) contrast with the relatively minor perturbations found for other disease-linked mutations analyzed previously that are also located within the same structural domain.

    “Furth ermore, the observed structural and thermodynamic alterations for all LPG-associated apoE3 mutants described here and in Georgiadou et al. (18) come in contrast to the relatively minor perturbations found for other disease-linked mutations analyzed previously that are also located within the same structural domain (34).”

    From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [34]

    The Introduction and Results (page numbers unavailable) identify R136S, R145C and K146E and report changes in their biophysical and thermodynamic properties. But the paper studies these variants as linked to type III hyperlipoproteinemia, not as LPG-associated mutants. The comparison with other mutations is attached to citation (34), not to Georgiadou et al. (18). (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • ApoE variants with lower thermodynamic stability and changes in conformational plasticity have been shown to have altered interdomain interactions and defective physiological functions.

    “ApoE variants with lower thermodynamic stability and changes in conformational plasticity have been shown to have altered interdomain interactions and defective physiological functions (19, 24 -28).”

    From Molecular Basis for Increased Risk for Late-onset Alzheimer Disease Due to the Naturally Occurring L28P Mutation in Apolipoprotein E4 (2014) · cites this paper as [27]

    The Results report altered stability and conformational behavior in apoE3 variants, and the Discussion proposes that some mutations perturb interdomain interactions (pages not specified). But stability was not uniformly lower, and the Discussion says further functional analysis is needed to establish the role of these changes in apoE3 dysfunction. The claim presents those proposed links and defective physiological function more definitively than this paper does. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • The observations could reflect reduced cellular uptake and lysosomal degradation, consistent with impaired receptor binding of APOE3Ch to receptors such as LDLR and HSPGs.

    “These observations could also reflect reduced cellular uptake and lysosomal degradation, consistent impaired receptor binding of APOE3Ch to its receptors such as LDLR and HSPGs [3, 16, 17]”

    From Astrocytic APOE3-Christchurch expression ameliorates brain amyloid-β pathology in 5xFAD mice (2026) · cites this paper as [16]

    The Introduction identifies apoE3[R136S] as the Christchurch variant and reports 60% reduced LDL-receptor binding for its lipid-bound form. It reports reduced HSPG binding for other apoE3 variants, not Christchurch, and does not address reduced cellular uptake or lysosomal degradation. Those parts of the proposed explanation are not supported by this paper. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Secondary-structure analysis method named inconsistently

    Minor

    The methods identify CDSSR as the selected method, but Table 1 labels the corresponding secondary-structure estimates as CDSSTR results.

1 of 1

Cites

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1 of the 77 references couldn’t be matched to a known paper.

Cited by

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Related

Authors
Georgiadou, D., Chroni, A., Vezeridis, A., Zannis, V. I., & Stratikos, E.
Affiliations
  • National Centre of Scientific Research "Demokritos" (GR)
  • Boston University (US)
Corresponding author
Stratikos, Efstratios
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-11-01
Volume
6
Issue
11
Pages
e27037
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0027037
PMC
PMC3206067
OpenAlex
W2087788439
Semantic Scholar
baff2bfc5283febb0fd938ea1cdc7cf39b7a7295
Cited by
24
References
77
Citations by year
2026: 3 · 2025: 2 · 2023: 1 · 2022: 1 · 2021: 2 · 2020: 1 · 2019: 1 · 2018: 2 · 2017: 1 · 2016: 1 · 2014: 4 · 2013: 2 · 2012: 3
FWCI
1.17
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • European Commission (037631)
  • Centre National de la Recherche Scientifique
  • National Institutes of Health (HL007969)
  • National Heart, Lung, and Blood Institute (HL 68216, HL-007969, R01HL068216, T32HL007969)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
  • Health Sciences › Medicine › Surgery › Lipoproteins and Cardiovascular Health
Keywords
apolipoprotein E3, type III hyperlipoproteinemia, N-terminal domain, protein stability, protein unfolding thermodynamics, protein secondary structure, protein oligomerization, hydrophobic surface exposure, LDL receptor binding
MeSH terms
Anilino Naphthalenesulfonates, Astrocytoma, Biophysics, Circular Dichroism, Fluorescent Dyes, Humans, Hyperlipoproteinemia Type III, Kinetics, Mutation, Protein Binding, Protein Conformation, Receptors, LDL, Recombinant Proteins, Tumor Cells, Cultured, Genetic Variation, Mutagenesis, Site-Directed, Protein Structure, Secondary, Apolipoprotein E3

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-31.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-31.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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