Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy
Open accessAbstract
Lipoprotein glomerulopathy (LPG) is a dominant inherited kidney disorder characterized by lipoprotein thrombi in glomerular capillaries. Single-amino-acid mutations in apoE have been associated with the development of the disease, although the mechanism is unknown. In an effort to gain mechanistic insight linking the presence of such mutations and the development of LPG, we evaluated the effects of three of the most common apoE3 variants associated with this disease, namely R145P(Sendai), R147P(Chicago), and R158P(Osaka or Kurashiki), on the structural and conformational integrity of the protein. All three variants were found to have significantly reduced helical content, to expose a larger portion of hydrophobic surface to the solvent, and to be significantly thermodynamically destabilized, often lacking functionally relevant unfolding intermediates. Furthermore, all variants were aggregation prone and had enhanced sensitivity to protease digestion. Finally, although the variants were able to form discoidal lipoprotein particles, discrete subpopulations of poorly formed or aberrant particles were evident. Furthermore, these lipoprotein particles were thermodynamically destabilized and aggregation prone. Overall, our data suggest that these mutations induce a generalized unfolding of the N-terminal domain of apoE3 toward a molten-globule-like structure. ApoE3 N-terminal domain unfolding due to mutation may constitute a common mechanism underlying the protein's association with the pathogenesis of LPG.
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Written from the paper’s full text.
TL;DR
The paper shows that three lipoprotein glomerulopathy-associated apoE3 mutations—R145P, R147P, and R158P—destabilize the N-terminal domain, causing loss of helicity, hydrophobic exposure, thermodynamic destabilization, aggregation, and protease sensitivity. It proposes that mutation-induced N-terminal unfolding toward a molten-globule-like state is a common mechanism linking these apoE3 variants to LPG pathogenesis.
Key findings
- All three variants had significantly reduced helical content, with R145P showing >10% loss, and all exposed more hydrophobic surface; ANS fluorescence enhancement was at least 50% for R147P and R158P.
- The variants were thermodynamically destabilized: thermal unfolding occurred at lower temperature with little cooperativity, losing 8–15 kcal/mol in apparent enthalpy; chemically, R145P had a second-transition apparent ΔG of 5.63 kcal/mol versus 10.1 kcal/mol for wild-type, R147P showed weak intermediate formation, and R158P showed no unfolding intermediate.
- All variants were aggregation-prone at 37°C, reaching hydrodynamic diameters up to the 100 nm range while wild-type remained stable, and all were more sensitive to elastase, trypsin, and chymotrypsin; DMPC remodeling slow phase was faster for variants.
- Reconstituted HDL-like particles containing variants were defective: wild-type particles averaged 11.9 nm diameter (distribution width 2.5 nm), R145P averaged 13.4 nm, R147P averaged 13.9 nm, and R158P formed poorly formed clusters; variant particles had lower helical content, thermal midpoints 8–9°C lower, and with PLA2, R145P- and R147P-containing particles became 100% aggregated after 24 h.
Methods
- Recombinant wild-type and mutant apoE3 (R145P, R147P, R158P) were expressed in *E. coli*, purified by Ni-NTA chromatography and 3C protease cleavage, refolded, and confirmed ≥98% pure by SDS-PAGE.
- Biophysical characterization included far-UV circular dichroism for secondary structure and thermal denaturation, guanidine hydrochloride chemical denaturation with tryptophan fluorescence, ANS fluorescence for hydrophobic exposure, DLS for aggregation, protease sensitivity assays with trypsin/chymotrypsin/elastase, DMPC remodeling kinetics, electron microscopy of HDL-like discoidal particles, and PLA2-induced aggregation.
- HDL-like particles were prepared at a POPC:cholesterol:apoE3:sodium cholate molar ratio of 100:10:1:100, and wild-type and variant particles were analyzed in parallel.
Limitations
- Stated: the thermal denaturation transition of apoE is not an ideal two-state model, so the reported enthalpy changes are apparent; chemical denaturation was fitted to a three-state model yielding apparent ΔG values.
- Evident: all experiments use recombinant apoE3 and reconstituted HDL-like particles in vitro, with no patient-derived lipoproteins, kidney tissue, or in vivo LPG model, so the causal link to glomerular lipoprotein deposition remains inferential.
- Evident: only three LPG-associated apoE3 variants were studied, and some particle morphology and proteolysis data appear in supplementary figures, so generalization to other apoE mutations or isoforms is not established.
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Claims the paper makes
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- Findingreasoning§ Mutations cause N-terminus misfolding
The misfolding of the N-terminal domain enhances the kinetics of interactions of apoE3 with phospholipid vesicles.
“Interestingly, the misfolding of the N-terminal domain actually enhances the kinetics of interactions of apoE3 with phospholipid vesicles”
Quote in the paperSubstantial claim - Findingown data§ Thermodynamic stability
The R145P mutant showed an unfolding profile similar to that of wild-type apoE3, including the intermediate stage.
“The R145P mutant showed an unfolding profi le similar to that of wild-type apoE3, including the intermediate stage ( Fig. 4A ).”
Quote in the paperSubstantial claim - Findingown data§ Thermodynamic stability
The R145P mutant had a lower apparent ΔG for the second unfolding transition (5.63 kcal/mol) than wild-type apoE3 (10.1 kcal/mol).
“However, the second unfolding transition was less cooperative and had a lower apparent 𝚫 G of 5.63 kcal/mol versus 10.1 kcal/mol for the wild-type.”
Quote in the paperSubstantial claim - Findingown data§ Protease sensitivity
The protease-resistant apoE3 species were absent from the R145P, R147P, and R158P digestion reactions.
“In all cases, the protease-resistant apoE3 species were absent from the R145P, R147P, and R158P digestion reactions”
Quote in the paperSubstantial claim - Findingown data§ Hydrophobic surface exposure
Enhancement of the ANS signal by at least 50% was evident for variants R147P and R158P.
“Enhancement of the signal by at least 50% was evident for variants R147P and R158P, suggesting a highly extensive exposure of hydrophobic core residues to the solvent that is consistent with partial unfolding of the N-terminal 4-helix bundle of apoE3.”
Quote in the paperSubstantial claim
- Findingown data§ Analysis of reconstituted HDL-like discoidal particles
Twenty-four hours of incubation with 100 ng/ml bee venom PLA2 resulted in only moderate aggregation of particles made with wild-type apoE3.
“Twenty-four hours of incubation with 100 ng/ml bee venom PLA 2 resulted in only moderate aggregation of particles made with wild-type apoE3.”
Quote in the paperSubstantial claim - Findingown data§ Thermodynamic stability
All three apoE3 variants showed a marked decrease in thermal stability, losing 8-15 kcal/mol in enthalpy change during denaturation.
“All three variants showed a marked decrease in thermal stability, losing 8-15 kcal/ mol in enthalpy change during denaturation ( Fig. 3D ).”
Quote in the paperSubstantial claim - Findingown data§ Analysis of reconstituted HDL-like discoidal particles
HDL-like particles containing apoE3 R145P or R147P showed slightly higher average diameters of 13.4 and 13.9 nm, respectively.
“HDL-like particles containing apoE3R145P or apoE3R147P showed slightly higher average diameters of 13.4 and 13.9 nm, respectively”
Quote in the paperNot a substantial claim - Findingown data§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy
All three apoE3 variants are aggregation prone.
“Furthermore, all variants were aggregation prone and had enhanced sensitivity to protease digestion.”
Quote in the paperSubstantial claim - Findingown data§ Thermodynamic stability
All three apoE3 variants displayed very little cooperativity of unfolding compared with wild-type apoE3.
“In all cases, the variants unfolded at a signifi cantly lower temperature and displayed very little cooperativity of unfolding compared with the wild-type, indicating signifi cant thermodynamic destabilization.”
Quote in the paperSubstantial claim - Findingown data§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy
All three apoE3 variants had enhanced sensitivity to protease digestion.
“Furthermore, all variants were aggregation prone and had enhanced sensitivity to protease digestion.”
Quote in the paperSubstantial claim - Findingown data§ Thermodynamic stability
A significant portion of the R145P, R147P, and R158P mutants is unfolded at physiological temperatures (37°C).
“It should be noted that the thermal denaturation profi les indicate that a signifi cant portion of the R145P, R147P, and R158P mutants is unfolded at physiological temperatures (37°C, Fig. 3A-C , gray line).”
Quote in the paperSubstantial claim - Findingown data§ Protease sensitivity
All three apoE3 variants (R145P, R147P, R158P) were overall more sensitive to proteolytic digestion than wild-type, regardless of the protease used.
“All three variants were found to be overall more sensitive to proteolytic digestion, regardless of the protease used.”
Quote in the paperSubstantial claim - Findingown data§ Mutations cause aggregation of apoE3 and structurally defective lipoprotein particles
All three apoE3 variants rapidly transitioned to higher aggregation levels upon incubation at 37°C.
“all three variants rapidly transitioned to higher aggregation levels upon incubation at 37°C.”
Quote in the paperSubstantial claim - Findingown data§ Thermodynamic stability
All three apoE3 variants unfolded at a significantly lower temperature than wild-type apoE3.
“In all cases, the variants unfolded at a signifi cantly lower temperature and displayed very little cooperativity of unfolding compared with the wild-type, indicating signifi cant thermodynamic destabilization.”
Quote in the paperSubstantial claim - Findingown data§ Aggregation state
Upon incubation at 37°C, all three apoE3 variants displayed larger hydrodynamic diameters, up to the 100 nm range, in contrast to wild-type protein, which was stable.
“However, upon incubation at 37°C, all three variants displayed larger hydrodynamic diameters, up to the 100 nm range, in contrast to the wild-type protein, which was stable ( Fig. 6 ).”
Quote in the paperSubstantial claim - Interpretationown data§ Hydrophobic surface exposure
All three apoE3 variants have a higher degree of hydrophobic area exposure than wild-type apoE3.
“All variants resulted in signifi cantly higher ANS fl uorescence and a shift in fl uorescence maximum toward lower wavelength, compared with wild-type ( Fig. 5B ), indicating a higher degree of hydrophobic area exposure in the mutated proteins.”
Quote in the paperSubstantial claim - Interpretationown data§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy
The findings provide structural insight for the causal link between these mutations and the pathogenesis of LPG.
“Our fi ndings provide structural insight for the causal link between these mutations and the pathogenesis of LPG.”
Quote in the paperSubstantial claim - Premisecitation§ Protein expression and purifi cation
Wild-type apoE produced in the bacterial expression system used has biophysical properties identical to apoE produced by mammalian expression systems.
“Wild-type apoE produced in this expression system has been shown to have biophysical properties identical to apoE produced by mammalian expression systems ( 39 ).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [39]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- Interpretationown data§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy
The data suggest that the mutations induce a generalized unfolding of the N-terminal domain of apoE3 toward a molten-globule-like structure.
“Overall, our data suggest that these mutations induce a generalized unfolding of the N-terminal domain of apoE3 toward a molten-globule-like structure.”
Quote in the paperSubstantial claim - Interpretationown data§ Analysis of reconstituted HDL-like discoidal particles
The structural and thermodynamic destabilization induced in apoE3 due to the introduction of the R145P, R147P, and R158P mutations also applies in HDL-like discoidal particles containing these apoE3 variants.
“These results, taken together, suggest that the structural and thermodynamic destabilization induced in apoE3 due to the introduction of the R145P, R147P, and R158P mutations also applies in HDL-like discoidal particles containing these apoE3 variants.”
Quote in the paperSubstantial claim - Interpretationreasoning§ Mutations cause aggregation of apoE3 and structurally defective lipoprotein particles
The misfolding of the N-terminal domain induced by the LPG-associated mutations had significant effects on the oligomerization state of apoE3.
“Accordingly, the misfolding of the N-terminal domain induced by the LPG-associated mutations had signifi cant effects on the oligomerization state of apoE3.”
Quote in the paperSubstantial claim - Interpretationreasoning§ Protease sensitivity
The changes in protease sensitivity indicate local unfolding that exposes new cleavage sites to the solvent.
“we conclude that the changes in protease sensitivity indicate local unfolding that exposes new cleavage sites to the solvent.”
Quote in the paperSubstantial claim - Interpretationown data§ Thermodynamic stability
All three apoE3 variants are significantly thermodynamically destabilized.
“In all cases, the variants unfolded at a signifi cantly lower temperature and displayed very little cooperativity of unfolding compared with the wild-type, indicating signifi cant thermodynamic destabilization.”
Quote in the paperSubstantial claim - Interpretationreasoning§ A causal link between apoE3 variants and LPG
N-terminal domain unfolding induced by mutation may constitute an important mechanism underlying the pathogenic potential of apoE.
“suggest that N-terminal domain unfolding induced by mutation may constitute an important mechanism underlying the pathogenic potential of apoE.”
Quote in the paperSubstantial claim - Interpretationreasoning§ Secondary structure
The reduction of helical content in the variants cannot be attributed solely to local perturbation of the helical structure due to the proline residue and suggests more extended, long-range perturbations in the folding of the protein.
“Such reduction of helical content cannot be attributed solely to local perturbation of the helical structure, due to the presence of the proline residue, and suggests more-extended, long-range perturbations in the folding of the protein.”
Quote in the paperSubstantial claim - Interpretationreasoning§ Analysis of reconstituted HDL-like discoidal particles
The PLA2-induced aggregation of lipoproteins containing these apoE3 variants may be physiologically relevant to the pathogenesis of LPG.
“it is possible that the PLA 2 -induced aggregation of lipoproteins containing these apoE3 variants may be physiologically relevant to the pathogenesis of LPG.”
Quote in the paperSubstantial claim- Rests on an assumption: Bee venom PLA2 is a valid model for secretory PLA2 in human kidney.
- Interpretationreasoning§ Mutations cause aggregation of apoE3 and structurally defective lipoprotein particles
The structural and thermodynamic destabilization of lipoprotein particles carrying the mutant apoE3 may contribute to aggregation and deposition in the kidney.
“the structural and thermodynamic destabilization of lipoprotein particles carrying the mutant apoE3 may contribute to aggregation and deposition in the kidney.”
Quote in the paperSubstantial claim
- May combine two claims. 24
- Classified as probably not a substantial claim. 9
May combine two claims.
Findingown data§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyThe lipoprotein particles formed with the variants were thermodynamically destabilized and aggregation prone.
“Furthermore, these lipoprotein particles were thermodynamically destabilized and aggregation prone.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data§ Analysis of reconstituted HDL-like discoidal particlesHDL-like particles made with wild-type apoE3 had an average hydrodynamic diameter of 11.9 nm and distribution width of 2.5 nm.
“the hydrodynamic diameter distribution of such lipoprotein particles made with wild-type apoE3 had an average diameter of 11.9 nm and distribution width of 2.5 nm”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown data§ Protein expression and purifi cationAll three apoE3 mutations studied (R145P, R147P, R158P) are located on helix 4 of the N-terminal moiety of the protein.
“All three apoE3 mutations are located on helix 4 of the N-terminal moiety of the protein ( Fig. 1A ).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown data§ Secondary structureAll three apoE3 variants maintained the overall CD spectrum shape characteristic of apoE, although small deviations for R158P were evident.
“All three variants maintained the overall spectrum shape characteristic of apoE, although small deviations for R158P were evident ( Fig. 2A ).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown data§ Analysis of reconstituted HDL-like discoidal particlesParticles made with apoE3 variants aggregated to a much higher degree, with particles containing R145P or R147P apoE3 becoming 100% aggregated after 24 h.
“In contrast, particles made with apoE3 variants, aggregated to a much higher degree, with particles containing R145P or R147P apoE3 becoming 100% aggregated after 24 h ( Fig. 10D ).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data§ Secondary structureAll three apoE3 variants had significantly reduced helical content compared with wild-type apoE3, with the loss especially pronounced for R145P (at least 10% loss).
“Calculation of the helical content of the variants indicated a signifi cant loss of helicity for all three variants that was especially pronounced for R145P ( H11601 10% loss) ( Fig. 2B ).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data§ Hydrophobic surface exposureAll three apoE3 variants resulted in significantly higher ANS fluorescence and a shift in fluorescence maximum toward lower wavelength compared with wild-type.
“All variants resulted in signifi cantly higher ANS fl uorescence and a shift in fl uorescence maximum toward lower wavelength, compared with wild-type ( Fig. 5B ), indicating a higher degree of hydrophobic area exposure in the mutated proteins.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data§ Thermodynamic stabilityThe R147P variant had an even less-cooperative transition, with very weak formation of an unfolding intermediate.
“The R147P variant had an even lesscooperative transition, with very weak formation of an unfolding intermediate ( Fig. 4B ).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data§ Thermodynamic stabilityThe R158P variant had a highly uncooperative transition, with no evidence for an unfolding intermediate.
“Finally, the R158P variant had a highly uncooperative transition, with no evidence for an unfolding intermediate ( Fig. 4C ).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyAlthough the variants were able to form discoidal lipoprotein particles, discrete subpopulations of poorly formed or aberrant particles were evident.
“Finally, although the variants were able to form discoidal lipoprotein particles, discrete subpopulations of poorly formed or aberrant particles were evident.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data§ Aggregation stateAll samples initially demonstrated an average hydrodynamic diameter of about 12 nm, with the exception of R158P, which had an average diameter of 14.3 nm.
“All samples initially demonstrated an average hydrodynamic diameter of about 12 nm, with the exception of R158P, which had an average diameter of 14.3 nm (width of distribution = 2.3 nm).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown data§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyReconstituted HDL-like discoidal particles containing these variants show structural and thermodynamic defects and can be prone to aggregation.
“Reconstituted HDL-like discoidal particles containing these variants show structural and thermodynamic defects and can be prone to aggregation.”
Quote in the paperSubstantial claimMay combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyAdenovirus-mediated gene transfer studies of apoE Sendai (R145P) in apoE-deficient mice resulted in distinct intra-glomerular depositions of apoE-containing lipoproteins, suggesting apoE Sendai is an etiological cause of LPG.
“adenovirus-mediated gene transfer studies of apoE Sendai (R145P) in apoEdefi cient mice resulted in distinct intra-glomerular depositions of apoE-containing lipoproteins, suggesting that apoE Sendai is an etiological cause of LPG ( 25 ).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [25]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
Interpretationown data§ Mutations cause N-terminus misfoldingThe mutations grossly interfere with the folding of the helical N-terminal domain of apoE3, with effects extending well away from the local area of the mutation to the thermodynamic behavior of the whole molecule.
“Our analysis suggests that the mutations grossly interfere with the folding of the helical N-terminal domain of apoE3, with effects extending well away from the local area of the mutation to the thermodynamic behavior of the whole molecule.”
Quote in the paperSubstantial claimMay combine two claims.
Premisecitation§ Mutations cause aggregation of apoE3 and structurally defective lipoprotein particlesLipid-free apoE is prone to oligomerization and forms poorly characterized dimeric, tetrameric, or octameric structures.
“Lipid-free apoE is prone to oligomerization and forms poorly characterized dimeric, tetrameric, or octameric structures ( 30, 50, 51 ).”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [30]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [50]
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- cites [51]
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). May combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyLipid-free apoE is folded to two seemingly independent structural domains that can be separated after digestion with thrombin, generating an N-terminal 22 kDa fragment and a C-terminal 10 kDa fragment.
“Lipid-free apoE is folded to two seemingly independent structural domains that can be separated after digestion with thrombin, generating an N-terminal 22 kDa fragment and a C-terminal 10 kDa fragment ( 29, 30 ).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [29]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [30]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Classified as probably not a substantial claim. May combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyA series of mutations within apoE have been closely associated with LPG and proposed to underlie disease pathogenesis, although the mechanism is unknown.
“A series of mutations within apoE, the majority of which are point mutations in which proline or cysteine is substituted for an arginine residue, have been closely associated with LPG and it has been proposed that they may underlie disease pathogenesis, although the mechanism is unknown ( 18-21 ).”
Quote in the paperSubstantial claim0 of 4 sources checked- cites [18]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [19]
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- cites [20]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [21]
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). May combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyApoE is prone to aggregation and to form oligomeric structures in solution.
“Furthermore, apoE has been shown to be prone to aggregation and to form oligomeric structures in solution ( 36, 37 ).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [36]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [37]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
Interpretationreasoning§ Mutations cause aggregation of apoE3 and structurally defective lipoprotein particlesSuch aggregation tendencies may contribute to the formation of abnormal lipoprotein particles and directly cause apolipoprotein or lipoprotein aggregates in the glomerular capillaries.
“Furthermore, such aggregation tendencies may contribute to the formation of abnormal lipoprotein particles and directly cause apolipoprotein or lipoprotein aggregates in the glomerular capillaries.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationcitation§ Thermodynamic stabilityThe second transition during chemical denaturation of apoE is suggested to be due to unfolding of the N-terminal region, and its destabilization or absence suggests that the N-terminal domains of the apoE3 variants are at least partially unfolded.
“The second transition during the chemical denaturation of apoE has been suggested to be due to the unfolding of the N-terminal region of the protein, and the destabilization or total absence of this transition suggests that the N-terminal domains of the apoE3 variants are at least partially unfolded ( 29, 32, 48 ).”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [29]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [32]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [48]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyApoE is highly helical with labile tertiary structure that can assume structures characteristic of a molten globule.
“ApoE is highly helical with labile tertiary structure that can assume structures characteristic of a molten globule ( 26 ).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26]
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).
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyThe N-terminal domain of apoE must unfold during lipid binding to allow for exposure of its hydrophobic core.
“Biophysical and computational analyses suggest that the N-terminal domain must unfold during lipid binding to allow for exposure of its hydrophobic core (27).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [27]
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).
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyThe N-terminal domain of apoE folds as a four-helix bundle of amphipathic alpha-helices in the lipid-free state.
“Structural analysis has indicated that the N-terminal domain folds as a four-helix bundle of amphipathic 𝛂 -helices in the lipid-free state ( 31 ).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [31]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
May combine two claims.
Interpretationown data§ Aggregation stateThe mutations severely affect the oligomerization properties of apoE and make the variants more aggregation-prone at physiological temperatures.
“We conclude that the mutations severely affect the oligomerization properties of apoE and make the variants more aggregation-prone at physiological temperatures.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyLPG is a dominant inherited disease with incomplete penetrance.
“LPG is a dominant inherited disease with incomplete penetrance ( 18 ).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [18]
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 conductown data§ Protein expression and purifi cationRecombinant wild-type and mutant apoE3 forms were purified to at least 98% purity as judged by SDS-PAGE.
“All proteins were H11601 98% pure as judged by SDS-PAGE analysis ( Fig. 1B ).”
Quote in the paperNot a substantial claimMay combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyApoE displays low thermodynamic stability and significant conformational plasticity, and mutations in the protein affect these properties, impeding physiological function.
“ApoE displays low thermodynamic stability and signifi cant conformational plasticity, and mutations in the protein have been shown to affect these properties, impeding physiological function ( 27, 32-35 ).”
Quote in the paperSubstantial claim0 of 5 sources checked- cites [27]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [32]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [33]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [34]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [35]
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). May combine two claims.
Premisecitation§ Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathyLPG is a rare kidney disorder that leads to progressive kidney failure and is characterized by lipoprotein thrombi in the glomerular capillaries.
“LPG is a rare kidney disorder that leads to progressive kidney failure and is characterized by lipoprotein thrombi in the glomerular capillaries ( 18, 19 ).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [18]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [19]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
The paper relies on this without support.
The aggregation-prone state of apoE3 variants is sufficient to form lipoprotein thrombi in the glomerular capillaries.
“It has been proposed that apoE mutations may display increased affi nity for the glomerulus and/or may be prone to aggregation favoring the formation of lipoprotein thrombi in the microenvironment of renal glomerulus ( 18 ).”
The paper relies on this without support.
Bee venom PLA2 is a valid model for secretory PLA2 in human kidney.
“We used bee venom PLA 2 to induce aggregation of HDL-like discoidal particles”
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?
Several LPG cases have been discovered due to high serum ApoE even without hyperlipidemia.
“In addition, several LPG cases have been discovered due to high serum ApoE even without hyperlipidemia [14, 15].”
From Membranous Nephropathy-Like Apolipoprotein E Deposition Disease with Apolipoprotein E Toyonaka (Ser197Cys) and a Homozygous Apolipoprotein E2/2 (2018) · cites this paper as [15]
The paper’s introductory discussion reports that most LPG patients have elevated plasma apoE, while elevated triglycerides and cholesterol occur only in some cases. This supports the observation that LPG can occur with high apoE without hyperlipidemia, but the paper does not say that high apoE causes LPG; it discusses apoE mutations as a possible cause. (LLM, conf=0.87)
Experimental — accuracy not yet measuredMany LPG pathogenic APOE mutations, such as APOE Kyoto (Arg25Cys), APOE Sendai (Arg145Pro), and APOE Chicago (Arg147Pro), significantly reduce the helicity of ApoE protein and increase its thermodynamic instability.
“Studies have found that many LPG pathogenic APOE mutations, such as APOE Kyoto (Arg25Cys), APOE Sendai (Arg145Pro), and APOE Chicago (Arg147Pro), significantly reduce the helicity of ApoE protein and increase its thermodynamic instability [17, 18].”
From A case report of youth-onset lipoprotein glomerulopathy with APOE Chicago mutation (2024) · cites this paper as [17]
The paper reports significantly reduced helical content and thermodynamic stability for apoE Sendai (R145P) and Chicago (R147P). Its study examined those variants and Osaka/Kurashiki (R158P), not Kyoto (R25C), so it does not support the claim about Kyoto. (LLM, conf=0.90)
Experimental — accuracy not yet measuredMutated ApoE-containing lipoproteins have higher aggregation tendency and are prone to interact with glomerular capillary endothelial cells and deposit in the glomerulus, which together lead to LPG pathogenesis.
“These mutated lipoproteins are with higher aggregation tendency and prone to interact with glomerular capillary endothelial cells and deposit in the glomerulus, which together lead to the pathogenesis of LPG.”
From A case report of youth-onset lipoprotein glomerulopathy with APOE Chicago mutation (2024) · cites this paper as [17]
The section “Mutations cause aggregation of apoE3 and structurally defective lipoprotein particles” (page unavailable) supports increased aggregation of mutant apoE-containing particles and proposes that this may contribute to kidney deposition. It does not establish an interaction with glomerular capillary endothelial cells; the paper says the mechanism of deposition is unknown and presents increased affinity for the glomerulus as a proposal, not a demonstrated cause of LPG. (LLM, conf=0.87)
Experimental — accuracy not yet measuredApoE 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 [28]
The Thermodynamic stability section reports that the studied apoE3 variants are less stable than wild-type, and Mutations cause N-terminus misfolding describes structural changes that perturb normal function (page numbers unavailable). The paper discusses interdomain interactions and proposes conformational changes between domains, but the cited passages do not show that these variants have altered interdomain interactions. (LLM, conf=0.82)
Experimental — accuracy not yet measuredThese structural changes led to defective binding capacity for the LDL receptor and enhanced protein and lipoprotein aggregation, which contribute to the pathogenesis of hyperlipoproteinemia and the formation of lipoprotein thrombi.
“These changes led to a defective binding capacity for the LDL receptor and enhanced protein and lipoprotein aggregation, which contribute to the pathogenesis of hyperlipoproteinemia and the formation of lipoprotein thrombi15).”
From The Novel Apolipoprotein E Mutation ApoE Chengdu (c.518T＞C, p.L173P) in a Chinese Patient with Lipoprotein Glomerulopathy (2018) · cites this paper as [15]
The section “Mutations cause aggregation of apoE3 and structurally defective lipoprotein particles” reports enhanced aggregation and suggests that it may contribute to kidney deposition and lipoprotein glomerulopathy; the paper also describes lipoprotein thrombi as a feature of that disease (page numbers unavailable). It does not establish defective LDL-receptor binding by these variants or show that the structural changes cause hyperlipoproteinemia. The proposed contribution to thrombus formation is more tentative than the citing sentence implies. (LLM, conf=0.87)
Experimental — accuracy not yet measured
Proteins with mutations causing LPG have a tendency to aggregate.
“Studies have shown that proteins with mutations causing LPG such as APOE Sendai, APOE Chicago, APOE Osaka/Kurashiki,40 APOE Kyoto, APOE Tsukuba, and APOE Las Vegas have a tendency to aggregate.41”
From Lipoprotein Glomerulopathy, First Case Report from Canada (2022) · cites this paper as [40]
The section “Mutations cause aggregation of apoE3 and structurally defective lipoprotein particles” reports that the three LPG-associated variants studied—Sendai, Chicago, and Osaka/Kurashiki—rapidly reached higher aggregation levels. The paper does not establish that the other variants listed in the citing sentence—Kyoto, Tsukuba, and Las Vegas—also tend to aggregate. (LLM, conf=0.86)
Experimental — accuracy not yet measuredThe substitution of proline with other amino acids is an important mutation, as it may lead to a decrease in α-helical content, exposure of hydrophobic surfaces, thermodynamic instability, and aggregation of ApoE, resulting in LPG.
“The substitution of proline with other amino acids is an important mutation, such as ApoE Sendai, ApoE Chicago, ApoE Chengdu, ApoE Guangzhou, and ApoE Osaka/Kurashiki, as it may lead to a decrease in α-helical content, exposure of hydrophobic surfaces, thermodynamic instability, and aggregation of ApoE, resulting in LPG.[29]”
From A boy and his mother with lipoprotein glomerulopathy: Two case reports and literature review (2025) · cites this paper as [29]
The paper’s introductory passage and ‘Mutations cause N-terminus misfolding’ section describe arginine being replaced by proline, not proline being replaced by other amino acids. The ‘Secondary structure’ and ‘A causal link between apoE3 variants and LPG’ sections support the reported loss of helicity, destabilization, aggregation, and possible link to LPG, but the study examines Sendai, Chicago, and Osaka/Kurashiki—not Chengdu or Guangzhou. Page numbers were not available in our copy. (LLM, conf=0.89)
Experimental — accuracy not yet measuredThe apoE3 variants R145P, R147P, and R158P display major thermodynamic destabilization.
“We showed that apoE3 variants carrying these mutations [R145P (apoE Sendai ), R147P (apoE Chicago ), and R158P (apoE Osaka or apoE Kurashiki )] displayed major thermodynamic destabilization, structural perturbations, and increased hydrophobic surface exposure to the solvent and were aggregation prone (18).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [18]
R145P and R158P were analyzed in a previous study by DLS and were found to be oligomerized following incubation at 37°C for 24 h.
“which had been analyzed in a previous study by DLS and were likewise found to be oligomerized following their incubation at 37°C for 24 h (18).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [18]
ANS changes its fluorescence properties when interacting with hydrophobic sites on apoE.
“we used the hydrophobic reporter probe ANS, which changes its fluorescence properties when interacting with hydrophobic sites on apoE (18)(19)(20).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [18]
In a previous study, apoE3 variants where a proline residue substituted another residue led to secondary content alterations, thermodynamic destabilization, hydrophobic surface exposure, and aggregation of apoE3 due to secondary structure destabilization resulting from the incompatibility of proline in helical regions.
“In a previous study, we had suggested that apoE3 variants where a proline residue substituted another residue led to secondary content alterations, thermodynamic destabilization, hydrophobic surface exposure, and aggregation of apoE3 due to secondary structure destabilization resulting from the incompatibility of proline in helical regions (18).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [18]
The apoE3 variants R145P, R147P, and R158P are aggregation prone.
“We showed that apoE3 variants carrying these mutations [R145P (apoE Sendai ), R147P (apoE Chicago ), and R158P (apoE Osaka or apoE Kurashiki )] displayed major thermodynamic destabilization, structural perturbations, and increased hydrophobic surface exposure to the solvent and were aggregation prone (18).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [18]
The apoE3 variants R145P, R147P, and R158P display structural perturbations.
“We showed that apoE3 variants carrying these mutations [R145P (apoE Sendai ), R147P (apoE Chicago ), and R158P (apoE Osaka or apoE Kurashiki )] displayed major thermodynamic destabilization, structural perturbations, and increased hydrophobic surface exposure to the solvent and were aggregation prone (18).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [18]
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 [18]
The apoE3 variants R145P, R147P, and R158P have increased hydrophobic surface exposure to the solvent.
“We showed that apoE3 variants carrying these mutations [R145P (apoE Sendai ), R147P (apoE Chicago ), and R158P (apoE Osaka or apoE Kurashiki )] displayed major thermodynamic destabilization, structural perturbations, and increased hydrophobic surface exposure to the solvent and were aggregation prone (18).”
From Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy (2018) · cites this paper as [18]
Some etiology studies using APOE Sendai suggest that reduced receptor-binding activity and thermodynamic abnormalities may play important roles in the pathogenesis of LPG.
“Some etiology studies using APOE Sendai suggest that the reduced receptor-binding activity [12] and thermodynamic abnormalities [13] may play important roles in the pathogenesis of LPG.”
From Lipoprotein glomerulopathy with a novel apolipoprotein E variant, APOE Kanto (Asp 151dup) (2024) · cites this paper as [13]
Mutated ApoE is more prone to denaturation at physiological temperature and possesses significantly higher aggregation tendency.
“Mutated ApoE is more prone to denaturation at physiological temperature and possesses significantly higher aggregation tendency [17, 18].”
From A case report of youth-onset lipoprotein glomerulopathy with APOE Chicago mutation (2024) · cites this paper as [17]
Mutations on APOE3 (variant ε3) of LPG induce thermodynamic destabilization and enhanced aggregation.
“This finding is consistent with reports that mutations on APOE3 (variant ε3) of LPG induce thermodynamic destabilization and enhanced aggregation (Georgiadou et al., 2013; Katsarou, Stratikos, & Chroni, 2018).”
From Clinical and genetic analysis of lipoprotein glomerulopathy patients caused by <i>APOE</i> mutations (2020) · cites this paper as Georgiadou et al., 2013
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.
PLA2 concentration differs between methods and results
MinorThe methods specify 150 ng/ml bee venom PLA2 for the HDL-like particle aggregation experiment, whereas the results and Fig. 10 caption specify 100 ng/ml.
“0.1 mg/ml apoE3 in HDL-like particles was mixed with 150 ng/ml (1,956 U/mg) bee venom phospholipase A 2”
“Twenty-four hours of incubation with 100 ng/ml bee venom PLA 2 resulted in only moderate aggregation of particles made with wild-type apoE3.”
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Self-citationTopics in lipoprotein glomerulopathy: an overview
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Katsarou, M., Stratikos, E., & Chroni, A. (2018) · Journal of Lipid Research · doi:10.1194/jlr.m088732 · Cited by 22 · see citing works
Self-citationThe use of pharmacological chaperones in rare diseases caused by reduced protein stability
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A Case of Lipoprotein Glomerulopathy with apoE Chicago and apoE (Glu3Lys) Treated with Fenofibrate
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Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4
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Clinical and genetic analysis of lipoprotein glomerulopathy patients caused by <i>APOE</i> mutations
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ApoE2-DARPin fusion proteins enable selective RNA transfer to CD8 T cells by lipid nanoparticles
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A case of normolipidemic lipoprotein glomerulopathy due to the APOE Kyoto variant
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An Updated Review and Meta Analysis of Lipoprotein Glomerulopathy
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- Authors
- Georgiadou, D., Stamatakis, K., Efthimiadou, E. K., Kordas, G., Gantz, D., Chroni, A., & Stratikos, E.
- Affiliations
- National Centre of Scientific Research "Demokritos" (GR)
- Boston University (US)
- Corresponding author
- Chroni, Angeliki
- Stratikos, Efstratios
- Venue
- Journal of Lipid Research
- Publisher
- Elsevier BV
- Published
- 2013-01
- Volume
- 54
- Issue
- 1
- Pages
- 164-176
- ISSN
- 0022-2275 (ISSN-L)
- PMC
- PMC3520522
- OpenAlex
- W2167876575
- Semantic Scholar
- 50a4119c6c5c73aeea5481d466440e8866fcbbd6
- Cited by
- 37
- References
- 62
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 2 · 2023: 1 · 2022: 3 · 2021: 4 · 2020: 1 · 2019: 1 · 2018: 7 · 2017: 2 · 2016: 1 · 2015: 2 · 2014: 5 · 2013: 2
- FWCI
- 1.16
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Lipid Research (publishedVersion, cc-by)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- 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
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- APOE3, lipoprotein glomerulopathy, inherited mutations, protein destabilization, protein aggregation, alpha-helix content, hydrophobic surface exposure, lipoprotein particles, protease sensitivity
- MeSH terms
- Dimyristoylphosphatidylcholine, Humans, Kidney Diseases, Kidney Glomerulus, Lipoproteins, Cholesterol, HDL, Models, Molecular, Mutation, Peptide Hydrolases, Thermodynamics, Protein Structure, Secondary, Protein Structure, Quaternary, Apolipoprotein E3, Protein Multimerization, Protein Stability, Hydrophobic and Hydrophilic Interactions
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