Thermodynamic destabilization and aggregation propensity as the mechanism behind the association of apoE3 mutants and lipoprotein glomerulopathy

Classified as probably not a substantial claim. May combine two claims.

ThT fluorescence intensity is low in aqueous solution but increases upon binding of the probe to amyloid aggregates.

“ThT fluorescence intensity is low in aqueous solution, but it increases upon binding of the probe to amyloid aggregates”

• cites [39] Hatters, 2006, Amino-terminal domain stability mediates apolipoprotein E aggregation into neurotoxic fibrils, J. Mol. Biol. sourcePartly supported

  In “ApoE aggregates adopt certain amyloid-like properties but are distinctly unique” (page unavailable), the paper states that thioflavin T fluorescence increases upon binding to amyloid fibrils. It does not state that the dye’s fluorescence is low in aqueous solution without aggregates. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [40] Ramella, 2011, Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis, PLoS One. sourceSupported

May combine two claims.

ApoE displays low thermodynamic stability and significant conformational plasticity.

“Several biophysical studies have shown that apoE displays low thermodynamic stability and significant conformational plasticity, and allelic differences as well as single point mutations have been shown to affect protein conformation and to hinder physiological function”

• cites [13] Hatters, 2006, Apolipoprotein E structure: insights into function, Trends Biochem. Sci. sourceNot checked — no full text of the source
• cites [15] Frieden, 2017, A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions, Proc. Natl. Acad. Sci. USA. sourcePartly supported

  The Results and Discussion describes intrinsically disordered and flexible regions in apoE3 and says apoE can adopt different conformational forms, supporting significant conformational plasticity. It does not establish that apoE has low thermodynamic stability; the passage says the role of charged residues in stability is unclear. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [16] Chetty, 2017, Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry, Proc. Natl. Acad. Sci. USA. sourceNot checked — no full text of the source

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

ThT has been used previously to monitor the aggregation and amyloidogenic propensity of apolipoproteins.

“has been used previously to monitor the aggregation and amyloidogenic propensity of apolipoproteins (39,40)”

• cites [39] Hatters, 2006, Amino-terminal domain stability mediates apolipoprotein E aggregation into neurotoxic fibrils, J. Mol. Biol. sourceSupported
• cites [40] Ramella, 2011, Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis, PLoS One. sourceSupported

May combine two claims.

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).”

• cites [18] Georgiadou, 2013, Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy, J. Lipid Res. sourceSupported

May combine two claims.

ApoE2 is not expected to lead to LPG, explaining the distinct-to-LPG phenotype of glomerulopathy rarely reported in apoE2 homozygotes.

“Therefore, apoE2 is not expected to lead to LPG, explaining the distinct-to-LPG phenotype of glomerulopathy rarely reported to apoE2 homozygotes (41,42).”

• cites [41] Sakatsume, 2001, Novel glomerular lipoprotein deposits associated with apolipoprotein E2 homozygosity, Kidney Int. sourcePartly supported

  The Abstract says apoE2 homozygosity had not been reported in association with LPG, and the Discussion describes the siblings’ glomerulopathy as distinct from LPG. Those passages support the distinction, but they do not establish that apoE2 cannot lead to LPG or that this explains the phenotype; the Discussion treats its mechanism as uncertain. Page numbers are unavailable in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [42] Kawanishi, 2013, Glomerulopathy with homozygous apolipoprotein e2: a report of three cases and review of the literature, Case Rep. Nephrol. Urol. sourcePartly supported

  The Introduction and Discussion describe apoE2 homozygote glomerulopathy as rare and histologically distinct from LPG. But the Discussion also reports LPG lesions in patients with an apoE2 homozygote phenotype and suggests apoE2 homozygosity may contribute to LPG; it does not support ruling out an LPG role for apoE2. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

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).”

• cites [18] Georgiadou, 2013, Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy, J. Lipid Res. sourceSupported
• cites [19] Argyri, 2014, Molecular basis for increased risk for late-onset Alzheimer disease due to the naturally occurring L28P mutation in apolipoprotein E4, J. Biol. Chem. sourceSupported
• cites [20] Dafnis, 2016, The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific, Sci. Rep. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Very rare cases of glomerulopathy, different from LPG because they are characterized by marked glomerular foam cell infiltration, have been reported in homozygotes for the apoE2 isoform and in most cases have been associated with type III hyperlipoproteinemia.

“Very rare cases of glomerulopathy, which are different from that of LPG, because they are characterized by marked glomerular foam cell infiltration, have been reported in homozygotes for the apoE2 isoform and, in most of the cases, have been associated with type III hyperlipoproteinemia (41,42).”

• cites [41] Sakatsume, 2001, Novel glomerular lipoprotein deposits associated with apolipoprotein E2 homozygosity, Kidney Int. sourceSupported
• cites [42] Kawanishi, 2013, Glomerulopathy with homozygous apolipoprotein e2: a report of three cases and review of the literature, Case Rep. Nephrol. Urol. sourcePartly supported

  The Introduction and Discussion describe apoE2-homozygote glomerulopathy as very rare and distinguish its marked glomerular foam-cell infiltration from LPG. The Abstract reports type III hyperlipoproteinemia in all three new cases, but the passages do not establish that it was present in most of the previously reported cases. No page numbers are available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

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).”

• cites [18] Georgiadou, 2013, Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy, J. Lipid Res. sourceSupported

May combine two claims.

ApoE is prone to aggregation and forms oligomeric structures in solution.

“Furthermore, apoE has been shown to be prone to aggregation and to form oligomeric structures in solution (21,22).”

• Also stated in: § Aggregation propensity of WT and mutant apoE3 forms · boosted

  “ApoE is known to be prone to self-association and to form oligomeric structures at higher concentrations (35,37,38).”

• cites [21] Gau, 2011, Mass spectrometry-based protein footprinting characterizes the structures of oligomeric apolipoprotein E2, E3, and E4, Biochemistry. sourceSupported
• cites [22] Huang, 2011, Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization, Biochemistry. sourceSupported
• cites [35] Clément-Collin, 2006, The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure, Biophys. Chem. sourceNot checked — no full text of the source
• cites [37] Aggerbeck, 1988, Human apolipoprotein E3 in aqueous solution. II. Properties of the amino- and carboxyl-terminal domains, J. Biol. Chem. sourceNot checked — no full text of the source
• cites [38] Perugini, 2000, Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid, J. Biol. Chem. sourceSupported

May combine two claims.

Most LPG patients have been reported to present elevated plasma apoE levels, while normalization or reduction of apoE levels following various treatments results in remission or improvement of the disease.

“Most of the LPG patients have been reported to present elevated plasma apoE levels (5, 6, 23-25), while it has been observed that normalization or reduction of apoE levels following various treatments (e.g., lipid-lowering treatments with fenofibrate or protein A immunoadsorption) result in remission or improvement of the disease (24,25,47).”

• cites [5] Matsunaga, 2014, Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia, Clin. Exp. Nephrol. sourceNot checked — no full text of the source
• cites [6] Stratikos, 2014, A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy, Clin. Exp. Nephrol. sourceNot checked — no full text of the source
• cites [23] Saito, 2001, Role of apolipoprotein E variants in lipoprotein glomerulopathy and other renal lipidoses, Clin. Exp. Nephrol. sourceNot checked — no full text of the source
• cites [24] Hu, 2014, Hereditary features, treatment, and prognosis of the lipoprotein glomerulopathy in patients with the APOE Kyoto mutation, Kidney Int. sourceNot checked — no full text of the source
• cites [25] Xin, 2009, Successful treatment of patients with lipoprotein glomerulopathy by protein A immunoadsorption: a pilot study, Nephrol. Dial. Transplant. sourceNot checked — no full text of the source
• cites [47] Wu, 2018, The novel apolipoprotein E mutation ApoE Chengdu (c.518TC, p.L173P) in a Chinese patient with lipoprotein glomerulopathy, J. Atheroscler. Thromb. sourcePartly supported

  The Discussion (page unavailable) says serum apoE is usually elevated in LPG. The Clinical Course (page unavailable) reports that apoE fell into the stated normal range while the patient improved after fenofibrate treatment. The paper does not establish that lowering plasma apoE caused remission, and the passages found do not discuss protein A immunoadsorption. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

Allelic differences and single point mutations affect apoE conformation and hinder physiological function.

“Several biophysical studies have shown that apoE displays low thermodynamic stability and significant conformational plasticity, and allelic differences as well as single point mutations have been shown to affect protein conformation and to hinder physiological function”

• cites [13] Hatters, 2006, Apolipoprotein E structure: insights into function, Trends Biochem. Sci. sourceNot checked — no full text of the source
• cites [15] Frieden, 2017, A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions, Proc. Natl. Acad. Sci. USA. sourcePartly supported

  The Results and Discussion (page unavailable) report structural differences between apoE3 and apoE4 associated with their single-amino-acid difference at position 112, and link those differences to lipid-binding specificity. The Abstract (page unavailable) also reports functional differences between the isoforms. The paper does not establish the broader claim that single-point mutations generally hinder apoE’s physiological function; it describes altered function, and reports that a five-mutation form remained biologically active. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [16] Chetty, 2017, Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry, Proc. Natl. Acad. Sci. USA. sourceNot checked — no full text of the source

May combine two claims.

WT apoE3 is stable under incubation at 37 degrees C for 24 h, in contrast to the mutants.

“in contrast to the WT apoE3, which was stable (Fig. 8A-D).”

• AssumptionRests on an assumption: The refolded recombinant apoE3 variants adopt native-like conformations comparable to WT apoE3, so measured differences reflect the effects of the mutations.

May combine two claims.

All three apoE3 variants R25C, R114C, and A152D are aggregation-prone, as shown by dynamic light-scattering measurements and enhanced thioflavin T binding.

“Furthermore, all three apoE3 variants are aggregation-prone, as shown by dynamic light-scattering measurements and by their enhanced capacity to bind the amyloid probe thioflavin T.”

• AssumptionRests on an assumption: The refolded recombinant apoE3 variants adopt native-like conformations comparable to WT apoE3, so measured differences reflect the effects of the mutations.

May combine two claims.

All three apoE3 mutations R25C, R114C, and A152D result in significantly higher ANS fluorescence compared with WT apoE3.

“All apoE3 mutations resulted in significantly higher ANS fluorescence compared with WT apoE3 (Fig. 5A, B)”

• AssumptionRests on an assumption: The refolded recombinant apoE3 variants adopt native-like conformations comparable to WT apoE3, so measured differences reflect the effects of the mutations.

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

All mutations are located on the N-terminal moiety of apoE3.

“All mutations are located on the N-terminal moiety of apoE3 (Fig. 1A).”

Classified as probably not a substantial claim.

A series of apoE mutations have been associated with LPG development.

“A series of apoE mutations have been associated with LPG development.”

May combine two claims.

A link between the thermodynamic stability of apoE3 variants, their propensity to aggregate, and the formation of lipoprotein thrombi in the glomerulus suggests that stabilization of the apoE3 variants may hold possible therapeutic benefit.

“A link between the thermodynamic stability of apoE3 variants, their propensity to aggregate, and the formation of lipoprotein thrombi in the glomerulus suggests that the stabilization of the apoE3 variants may hold possible therapeutic benefit.”

Classified as probably not a substantial claim. May combine two claims.

All lipoprotein samples were prepared using the same phospholipid-cholesterol suspension, and the procedure was performed in parallel.

“All lipoprotein samples were prepared using the same phospholipid-cholesterol suspension, and the procedure was performed in parallel.”

Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

Our results, combined with a previous study, provide a unified framework for understanding the potential pathogenic mechanism of these mutations and suggest a putative pathway for the discovery of small-molecule therapeutics for this disease.

“Our results, combined with a previous study, provide a unified framework for understanding the potential pathogenic mechanism of these mutations and suggest a putative pathway for the discovery of small-molecule therapeutics for this disease.”

May combine two claims.

Although liver is the major source of apoE in the circulation, apoE is also synthesized by the kidney, with a substantial portion of kidney protein synthesis committed to apoE synthesis.

“Although liver is the major source of apoE in the circulation, apoE is also synthesized by the kidney, with a substantial portion of kidney protein synthesis committed to apoE synthesis (43).”

• cites [43] Blue, 1983, Apolipoprotein E synthesis in human kidney, adrenal gland, and liver, Proc. Natl. Acad. Sci. USA. sourceNot checked — no full text of the source

Classified as probably not a substantial claim. The quote only partly states this claim.

All apoE3 forms were refolded and kept in a reducing environment by addition of 1 mM DTT to avoid oxidation of cysteine residues.

“Proteins were refolded and kept in a reducing environment by the addition of 1 mM DTT, to avoid oxidation of cysteine residues.”

May combine two claims.

Animal studies show that virus-mediated transduction of apoE2 in apoE−/− mice does not promote LPG-like intraglomerular depositions, in contrast to LPG-like depositions following apoE Sendai virus injection.

“This is supported further by animal studies showing that virus-mediated transduction of apoE2 in apoE / mice does not promote LPG-like intraglomerular depositions, in contrast to the presence of LPG-like depositions following apoE Sendai virus injection (26).”

• cites [26] Ishigaki, 2000, Virus-mediated transduction of apolipoprotein E (ApoE)-sendai develops lipoprotein glomerulopathy in ApoE-deficient mice, J. Biol. Chem. sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

Elevated apoE levels have been associated with the onset of glomerulus diseases in rats, and it has been proposed that apoE might be a risk factor for glomerulus lesions and renal disease.

“Elevated apoE levels have been associated with the onset of glomerulus diseases in rats (44,45), and it has been proposed that apoE might be a risk factor for glomerulus lesions and renal disease (46).”

• cites [44] van Goor, 1993, Renal apolipoproteins in nephrotic rats, Am. J. Pathol.Not checked — no full text of the source
• cites [45] Calandra, 1981, Secretion of lipoproteins, apolipoprotein A-I and apolipoprotein E by isolated and perfused liver of rat with experimental nephrotic syndrome, Biochim. Biophys. Acta. sourceNot checked — no full text of the source
• cites [46] Zhou, 2013, Signaling pathways of apoE and its role of gene expression in glomerulus diseases, J. Recept. Signal Transduct. Res. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The refolded recombinant apoE3 proteins were approximately 98% pure, as estimated by SDS-PAGE.

“The refolded proteins were approximately 98% pure, as estimated by SDS-PAGE.”

May combine two claims.

The mutations severely affect the oligomerization properties of apoE3 and make the mutant proteins more aggregation-prone at physiological temperatures.

“The findings suggest that the mutations severely affect the oligomerization properties of apoE3 and make the mutant proteins more aggregation-prone at physiological temperatures.”

• AssumptionRests on an assumption: The refolded recombinant apoE3 variants adopt native-like conformations comparable to WT apoE3, so measured differences reflect the effects of the mutations.

Classified as probably not a substantial claim.

All analyses were performed on freshly refolded proteins.

“All analyses were performed on freshly refolded proteins.”

May combine two claims.

ApoE2, which harbors the R158C substitution, has similar α-helical content as apoE3 and appears to be more thermodynamically stable compared with apoE3.

“ApoE2, which harbors the R158C substitution, has similar -helical content as apoE3 and appears to be more thermodynamically stable compared with apoE3 (36).”

• cites [36] Morrow, 2000, Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain, Biochemistry. sourceNot checked — no full text of the source

May combine two claims.

Specific substitutions in the N-terminal moiety of apoE3 that lead to secondary structure content changes, thermodynamic destabilization, and hydrophobic surface exposure have the potential of being pathogenic and point to a unifying mechanism linking apoE3 mutations and the pathogenesis of LPG.

“The two studies interpreted together rather suggest that specific substitutions in the N-terminal moiety of apoE3 that lead to secondary structure content changes, thermodynamic destabilization, and hydrophobic surface exposure have the potential of being pathogenic and thus point to a unifying mechanism linking apoE3 mutations and the pathogenesis of LPG.”

Classified as probably not a substantial claim.

The increased levels of plasma apoE in LPG patients along with increased mechanical pressure native to the glomerulus during normal filtration that leads to additional elevation of local apoE levels could enhance the aggregation kinetics of destabilized apoE mutants already susceptible to aggregation.

“The increased levels of plasma apoE in LPG patients along with the increased mechanical pressure native to the glomerulus during the normal filtration process that leads to additional elevation of the local apoE levels could enhance the aggregation kinetics of destabilized apoE mutants already susceptible to aggregation.”

• AssumptionRests on an assumption: The increased mechanical pressure native to the glomerulus during normal filtration leads to additional elevation of local apoE levels.

Classified as probably not a substantial claim.

Successful mutagenesis was confirmed by DNA sequencing.

“Successful mutagenesis was confirmed by DNA sequencing.”

May combine two claims.

The reduction of alpha-helical content cannot solely be attributed to local perturbation of the helical structure due to the mutated residue and suggests that all mutations may exert more extended perturbations to local or global folding.

“Such reduction of -helical content cannot solely be attributed to local perturbation of the helical structure due to the presence of the mutated residue and suggests that all mutations may exert more extended perturbations to the local or global folding of the protein.”

• AssumptionRests on an assumption: The refolded recombinant apoE3 variants adopt native-like conformations comparable to WT apoE3, so measured differences reflect the effects of the mutations.