Utilizing a cell culture based novel cellular thermal shift assay to understand the isoform-dependent thermal stability of ApoE variants

The arginine at position 112 in ApoE4 is predicted to promote an interaction between the N- and C-terminal domains (domain interaction) that is absent in ApoE3 and ApoE2.

“The arginine at position 112 in ApoE4 is predicted to promote an interaction between the N-and C-terminal domains (domain interaction) that is absent in ApoE3 and ApoE2 23,26,27 .”

• cites [23] sourceSupported
• cites [26] sourceNot checked — no full text of the source
• cites [27] 2020, Apolipoprotein E4 exhibits intermediates with domain interaction, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics sourceNot checked — no full text of the source

APOE4-R251G decreases the risk of ApoE4 to be similar to that of ApoE3.

“ApoE4-R251G has been shown to decrease the risk of ApoE4 to be similar to that of ApoE3 37,38”

• cites [37] 2022, Association of Rare APOE Missense Variants V236E and R251G With Risk of Alzheimer Disease, JAMA Neurol sourcePartly supported

  The Discussion reports that R251G mitigates the Alzheimer disease risk associated with APOE ε4. But it says the resulting risk estimate is similar to APOE ε2, specifically ε2/ε3—not ε3 as the citing sentence states. The Results likewise compare the ε3/ε4 (R251G) group with ε2/ε3. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [38] sourcePartly supported

  The introductory passage labeled Abstract (page unavailable) says R251G has been associated with reduced Alzheimer disease risk. It does not say the reduction occurs specifically on an ApoE4 background or brings risk to the level of ApoE3; the paper’s reported analysis concerns R145C instead. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

APOE3-R145C is associated with increased risk of AD compared with ApoE3.

“APOE3-R145C has been shown to increase the risk of AD 38 .”

• Also stated in: § Testing the thermal stability of rare disease-associated ApoE variants · plain

  “ApoE3-R145C has been associated with increased risk when compared with ApoE3”

• cites [38] sourcePartly supported

  The Results and Discussion report increased Alzheimer disease risk associated with APOE3-R145C in African-ancestry individuals with the ε3/ε4 genotype. The citing sentence omits that restriction: the paper reports no significant increase among ε3/ε3 carriers, so it does not support a general increase in risk compared with ApoE3. (LLM, conf=0.91)

  Experimental — accuracy not yet measured

The region around R25C is structurally similar between ApoE3 and ApoE4.

“The parallel effect of R25C 51 across isoforms suggests that this region is structurally similar between ApoE3 and ApoE4”

• cites [51] sourceCould not verify

  The Abstract reports structural effects of R25C in apoE3, and the Fig. 1 caption maps residue 25 on an apoE3 structure. Neither passage compares the region around R25C in apoE3 and apoE4; a search for parallel effects across isoforms found no such comparison in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

ApoE3-Jacksonville caused a significant reduction in the width of the slope.

“This corresponded to a similarly significant reduction in the width of the slope, which could be due to the reduced propensity of ApoE3-Jacksonville to dimerize or oligomerize 36 (Figure 3E)[ F (5, 35) = 7.419 (Dunnets ApoE3 vs ApoE3_Jacksonville p=0.0142,ApoE3 vs ApoE4 p=0.0056).”

• cites [36] sourceSupported

Lipid-free ApoE readily self-associates through hydrophobic interactions, forming oligomers and aggregates that complicate thermal stability measurements.

“Lipid-free ApoE readily self-associates through hydrophobic interactions, forming oligomers and aggregates that complicate thermal stability measurements 40,41 .”

• cites [40] sourcePartly supported

  The Results (page unavailable) state that apoE forms a mixture of oligomers that hindered structural determination, and identify exposed hydrophobic residues in apoE3. They do not say that hydrophobic interactions cause self-association or aggregation, or that these processes complicate thermal stability measurements; the reported difficulty concerns NMR structure determination. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [41] Nemergut, M. et al. Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer’s Disease drug candidate. Mol Neurodegener 18, 38 (2023). sourcePartly supported

  The Background reports that lipid-free ApoE forms monomers, dimers, tetramers and octamers, and the Results link self-association to aggregation. But the Results describe the dimer interactions as primarily electrostatic, not hydrophobic. The Methods describe measuring thermal unfolding and aggregation together, but do not say aggregation complicates thermal-stability measurements; no page numbers are available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

In BiTSA, the complemented NanoLuc luciferase produces a luminescent signal proportional to the amount of soluble, properly folded ApoE.

“Upon cell lysis in the presence of LgBiT, the complemented NanoLuc luciferase produces a luminescent signal proportional to the amount of soluble, properly folded ApoE.”

• cites [45] sourceCould not verify

  The Materials and Methods section on fMRI Acquisition and Analysis describes brain imaging and functional connectivity, not a protein assay. Searches for BiTSA, NanoLuc, LgBiT, and soluble ApoE found no passage addressing the claimed luminescent signal. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: The N-terminal HiBiT tag does not alter the thermal stability or behavior of ApoE variants.

Mutations in the N-terminal receptor binding region likely act via mechanisms other than altered protein stability, such as heparan sulfate binding.

“suggesting that mutations in the N-terminal receptor binding region likely act via mechanisms other than altered protein stability, such as heparan sulfate binding 35 .”

• cites [35] sourcePartly supported

  The paper’s Abstract (page unavailable) places the APOE3 Christchurch R136S mutation in a receptor- and heparan-sulfate-proteoglycan-binding region and reports reduced heparin binding. It suggests that modulating APOE–HSPG interactions could reproduce a potentially protective effect. It does not assess protein stability or establish that mutations in this region generally act through mechanisms other than altered stability. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Lipid-free ApoE readily self-associates through hydrophobic interactions.

“Lipid-free ApoE readily self-associates through hydrophobic interactions, and this aggregation-prone behavior can complicate the interpretation of thermal stability data 41,63 .”

• cites [41] Nemergut, M. et al. Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer’s Disease drug candidate. Mol Neurodegener 18, 38 (2023). sourcePartly supported

  The Discussion (page not provided) proposes dimers as basic units of lipid-free ApoE aggregates, supporting self-association. But the Results section on the self-association interface (page not provided) says the modeled dimer interactions were formed primarily by electrostatic contacts, not hydrophobic interactions. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [63] sourcePartly supported

  The Results section (‘All three ApoE isoforms show similar size and shape’; page unavailable) reports that recombinant ApoE2, E3 and E4 form tetramers in solution. The Results section on ApoE4 assembly reports rapid further self-assembly for ApoE4, but much less for ApoE2 and ApoE3. The copy does not establish that hydrophobic interactions drive this association, so it supports the self-association point but not the stated mechanism. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Lipidation of ApoE protects it from aggregation.

“recent studies have shown that lipidation of ApoE protects it from aggregation 31 .”

• cites [31] sourceSupported

Post-translational modifications present in cells but absent in recombinant protein may influence the structural consequences of the L28P mutation.

“Second, post-translational modifications present in cells but absent in recombinant protein may influence the structural consequences of the L28P mutation 60,61 .”

• cites [60] sourcePartly supported

  The Discussion says O-glycosylation can affect protein structure and that APOE may be more heavily glycosylated inside cells than after secretion. It does not compare cellular APOE with recombinant protein or address the structural consequences of the L28P mutation. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [61] Lawler, P. E. et al. Apolipoprotein E O-glycosylation is associated with amyloid plaques and APOE genotype. Analytical Biochemistry 672, 115156 (2023). sourcePartly supported

  The Introduction (page unavailable) says apoE modifications are cell- and tissue-specific, and the Discussion (page unavailable) suggests that studying its glycoforms may clarify structural relationships. The paper does not address L28P or compare cellular with recombinant protein, so it does not support the proposed effect on that mutation's structural consequences. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Monomeric ApoE4 adopts conformations distinct from those observed in oligomeric forms.

“recent single-molecule studies have shown that monomeric ApoE4 adopts conformations distinct from those observed in oligomeric forms 24 .”

• cites [24] sourceSupported

Thermal stability may be a particularly sensitive indicator of structural perturbation in the helical bundle and domain-domain interaction regions.

“suggests that thermal stability may be a particularly sensitive indicator of structural perturbation in these regions 22,24,33,41,51 .”

• cites [22] sourceCould not verify

  The passage headed “Influence of Binding to Phospholipid on Domain Interaction” reports conformational changes measured by spin-label spectroscopy, and the Discussion describes changes in distance between domains. A separate passage mentions prior denaturation studies, but none says thermal stability is a particularly sensitive indicator of structural perturbation. Searches for thermal stability, melting temperature, and thermal unfolding did not locate that point in our copy; page numbers were unavailable. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [24] sourcePartly supported

  The Results section, “Folding and Stability of the Four-Helix Bundle” (page not available), relates measured stability to changes in the helical bundle; the Discussion, “Folding Equilibrium of Lipid-Free ApoE4” (page not available), also describes associated changes in the hinge and terminal regions. But the paper measures unfolding through chemical denaturant titration, not thermal stability, and does not establish thermal stability as a particularly sensitive indicator of perturbation. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [33] sourceNot checked — no full text of the source
• cites [41] Nemergut, M. et al. Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer’s Disease drug candidate. Mol Neurodegener 18, 38 (2023). sourcePartly supported

  The Discussion (page unavailable) links ApoE4’s altered intradomain interactions and H3-helix conformation to greater susceptibility to thermal denaturation than ApoE3 and ApoE2. The Methods describe measuring thermal unfolding. The paper does not establish that thermal stability is a particularly sensitive indicator of perturbations in both the helical bundle and domain–domain interaction regions. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [51] sourcePartly supported

  The section on thermodynamic stability of lipid-free apoE3 reports altered thermal unfolding in mutants with secondary-structure perturbations, and the lipoprotein-particle section says perturbed thermal profiles may indicate conformational defects. The paper does not establish that thermal stability is *particularly sensitive* relative to other indicators or specifically localize the detected perturbations to domain–domain interaction regions. Page numbers are unavailable in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The aggregation-prone behavior of ApoE can complicate the interpretation of thermal stability data.

“Lipid-free ApoE readily self-associates through hydrophobic interactions, and this aggregation-prone behavior can complicate the interpretation of thermal stability data 41,63 .”

• cites [41] Nemergut, M. et al. Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer’s Disease drug candidate. Mol Neurodegener 18, 38 (2023). sourcePartly supported

  The Background describes lipid-free ApoE as self-associating, and the Results report greater aggregation of ApoE4 than ApoE3. The Methods say the thermal-denaturation assay monitored unfolding and aggregation separately, but our copy does not say that aggregation complicates interpretation of thermal stability data. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [63] sourcePartly supported

  The Discussion (page unavailable) links ApoE4’s measured loss of stability under high-temperature conditions to its propensity to polymerize, and the Thermal denaturation methods report irreversible unfolding. These passages support a connection between aggregation and thermal-stability measurements, but do not explicitly say that aggregation complicates interpretation of the data. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

ApoE has a well-documented tendency to self-associate and form oligomers.

“ApoE presents particular challenges due to its well-documented tendency to self-associate and form oligomers 40,62 .”

• cites [40] sourceSupported
• cites [62] sourceSupported

Our findings appear to contrast with previous work reporting that L28P destabilizes ApoE4 in studies using purified recombinant protein.

“Our findings appear to contrast with previous work by 58,59 ,who reported that L28P destabilizes ApoE4 in studies using purified recombinant protein.”

• cites [58] sourceSupported
• cites [59] 2018, Structural Biology Helps Interpret Variants of Uncertain Significance in Genes Causing Endocrine and Metabolic Disorders, J Endocr Soc sourcePartly supported

  The Apolipoprotein E case description (§3.B; page unavailable) reports that ApoE4 Leu28Pro was studied in vitro and that the mutant protein was thermodynamically unstable. It does not specify purified recombinant protein, and it presents the experimental finding as a report from an earlier study rather than its own result. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

E96K showed no impact on thermal stability in ApoE3.

“E96K showed no impact on the thermal stability in ApoE3 although it showed significant destabilization of ApoE4 [Dunnets ApoE4 vs ApoE4_E96K p<0.0001]”

ApoE3-Christchurch had no impact on thermal stability.

“while ApoE3-Christchurch and ApoE3-R145C had no impact on the thermal stability”

In AlphaFold models, ApoE4-L28P shows the helical structure in this region is largely restored in four of five models.

“In contrast, the ApoE4-L28P model (Figure 4E) shows that the helical structure in this region is largely restored (again in four of five models)”

• AssumptionRests on an assumption: AlphaFold models accurately predict the structural consequences of L28P in ApoE3 and ApoE4.

In HEK293 cells, ApoE4 consistently exhibited a lower Tm than ApoE3 in BiTSA.

“ApoE4 consistently exhibited a lower Tm than ApoE3 (Figure 2B-C)[ F (4, 24) = 125.3,(Tukey's post-hoc ApoE3 to ApoE4 p<0.0001)].”

E96K showed significant destabilization of ApoE4.

“E96K showed no impact on the thermal stability in ApoE3 although it showed significant destabilization of ApoE4 [Dunnets ApoE4 vs ApoE4_E96K p<0.0001]”

The delta of the stability difference between ApoE3 and ApoE4 in BiTSA is approximately 2-3°C.

“The delta of the stability difference between ApoE3 and ApoE4 (~2-3°C) was consistent with previous reports using purified recombinant protein 28,32”

• cites [28] sourceNot checked — no full text of the source
• cites [32] sourceNot checked — no full text of the source

ApoE4-R251G caused a slight but significant increase in thermal stability compared with ApoE4.

“ApoE4-R251G, however, caused a slight but significant increase in the thermal stability when compared with ApoE4 (Dunnets ApoE4 vs ApoE4_R251G p=0.0003) (Figure 3C-D).”

Introduction of L28P into ApoE3 resulted in significant destabilization.

“Introduction of L28P into ApoE3 resulted in significant destabilization, consistent with the expected disruption of helical structure (Figure 4C) [Dunnets ApoE3 vs ApoE3_L28P p<0.0001].”

ApoE3-Jacksonville caused a slight destabilization of ApoE3.

“ApoE3-Jacksonville caused a slight destabilization of the protein (Figure 3C-D)[ F(5, 37) = 28.98 (Dunnett's ApoE3 vs ApoE3_Jacksonville p=0.0446)].”

R25C caused modest destabilization when introduced into both the ApoE3 and ApoE4 backbone.

“R25C caused modest destabilization when introduced into both the ApoE3 and the ApoE4 backbone (Figure 4B-C)”

In AlphaFold models, ApoE3-L28P introduces a pronounced kink in helix 1 in four of five predicted models, disrupting regular helical geometry.

“In the ApoE3-L28P model (Figure 4D), the proline substitution introduces a pronounced kink in helix 1 in four of five predicted models, disrupting the regular helical geometry and potentially destabilizing inter-helical packing within the four-helix bundle.”

• AssumptionRests on an assumption: AlphaFold models accurately predict the structural consequences of L28P in ApoE3 and ApoE4.

ApoE3-R145C had no impact on thermal stability.

“while ApoE3-Christchurch and ApoE3-R145C had no impact on the thermal stability”

Introduction of L28P into ApoE4 resulted in paradoxical stabilization.

“However, when L28P was introduced into ApoE4, we observed a paradoxical stabilization of the protein (Figure 4B)[Dunnets ApoE4 vs ApoE4_L28P p<0.0001].”

The stability differences between ApoE isoforms were maintained in immortalized human astrocytes.

“The stability differences between isoforms were maintained in this glial cell context, supporting the use of BiTSA as a reliable method for assessing ApoE thermal stability (Figure 2D-E).”

ApoE4 exhibits significantly reduced thermal stability compared to ApoE3 in native brain tissue, including humanized APOE knock-in mouse brain and post-mortem human brain.

“We observed that ApoE4 exhibited a significantly lower melting temperature (Tm) compared to ApoE3, indicating reduced thermal stability of the ApoE4 isoform in the context of brain tissue (Figure 1A-B”

• Also stated in: § Thermal stability of ApoE isoforms in brain tissue · plain

  “human ApoE4 displayed reduced thermal stability compared to ApoE3 (Figure 1C-D)[ t=2.071, df=7, p=0.0386].”

• Also stated in: § Discussion · boosted

  “Our findings confirm that ApoE4 exhibits reduced thermal stability compared to ApoE3 in both mouse and human brain tissue, consistent with previous biophysical studies using purified recombinant protein 28,32,33 .”

In both mouse brain and mouse immortalized astrocytes, the Tms were approximately 1°C lower than the corresponding human context.

“It is intriguing that in the context of both mouse brain and mouse immortalized astrocytes the Tms were ~1°C lower than the corresponding human context.”

Our findings reveal complex relationships between ApoE sequence, isoform background, and thermal stability.

“Our findings reveal complex relationships between ApoE sequence, isoform background, and thermal stability that have implications for understanding the structural basis of ApoE-associated disease risk.”

AlphaFold model of ApoE4-L28P shows restoration of helical structure.

“AlphaFold model of ApoE4-L28P showing restoration of helical structure; cyan represents native ApoE4 and blue represents ApoE4-L28P.”

• AssumptionRests on an assumption: AlphaFold-predicted structures accurately reflect the structural consequences of the L28P mutation in ApoE3 and ApoE4.

Certain mutations have isoform-dependent effects.

“The isoform-dependent effects of certain mutations highlight the importance of considering genetic background when evaluating the consequences of rare variants, both for mechanistic understanding and for clinical interpretation of genetic findings.”

AlphaFold model of ApoE3-L28P shows disruption of helix 1 structure.

“AlphaFold model of ApoE3-L28P showing disruption of helix 1 structure; pink represents native ApoE3 and dark red represents ApoE3-L28P.”

• AssumptionRests on an assumption: AlphaFold-predicted structures accurately reflect the structural consequences of the L28P mutation in ApoE3 and ApoE4.

Mice with different humanized ApoE isoforms show different melting points (V50).

“Mice with different humanized ApoE isoforms show different melting points (V50, the temperature at which 50% of protein remains soluble, indicated next to model name).”

ApoE4 exhibits reduced thermodynamic stability compared to ApoE3 in prior studies using purified recombinant protein.

“ApoE4 exhibits reduced thermodynamic stability compared to ApoE3, but prior characterisation has relied on purified recombinant protein”

• Also stated in: § Introduction · boosted

  “Structural studies have revealed that ApoE4 exhibits reduced thermodynamic stability compared to ApoE3, with the four-helix bundle of ApoE4 being more susceptible to denaturation 28,32,33 .”

• cites [28] sourceNot checked — no full text of the source
• cites [32] sourceNot checked — no full text of the source
• cites [33] sourceNot checked — no full text of the source

The L28P mutation may have different structural consequences depending on the pre-existing helical architecture of each isoform.

“The L28P mutation may therefore have different structural consequences depending on the pre-existing helical architecture of each isoform.”

Thermal stability does not directly correlate with disease risk status.

“Collectively, these results indicate that thermal stability does not directly correlate with disease risk status”

The region surrounding position 112 differs between ApoE3 and ApoE4.

“whereas the divergent effects of R114C highlight differences in the region surrounding position 112.”

The structural context provided by the ApoE4 backbone, particularly the domain interaction, may compensate for or even benefit from the conformational changes induced by the proline substitution.

“This unexpected finding suggests that the structural context provided by the ApoE4 backbone, particularly the domain interaction, may compensate for or even benefit from the conformational changes induced by the proline substitution.”

BiTSA allows rapid, quantitative measurement of protein thermal stability in living cells.

“we developed a bioluminescence-based thermal stability assay (BiTSA) that allows rapid, quantitative measurement of protein thermal stability in living cells.”

The stability differences between ApoE isoforms are maintained in the complex cellular environment where ApoE exists in association with lipids and other binding partners.

“Importantly, we show that these stability differences are maintained in the complex cellular environment where ApoE exists in association with lipids and other binding partners.”

The thermal stability differences between ApoE isoforms observed with purified proteins are maintained in the complex environment of brain tissue, where ApoE exists in association with lipids and other binding partners.

“These results establish that the thermal stability differences between ApoE isoforms observed with purified proteins are maintained in the complex environment of brain tissue, where ApoE exists in association with lipids and other binding partners.”

Amino acid substitutions have differential effects depending on the local environment of the protein and the potential domain-domain interactions interrupted.

“These divegent responses suggest that amino acid substitutions have differential effects depending on the local environment of the protein and the potential domain-domain interactions interrupted.”

Structural changes that do not alter aggregation temperature would not be detected by thermal stability assays.

“as structural changes that do not alter aggregation temperature would not be detected.”

This study establishes BiTSA as a robust and scalable approach for assessing the thermal stability of ApoE variants in a cellular context.

“In conclusion, this study establishes BiTSA as a robust and scalable approach for assessing the thermal stability of ApoE variants in a cellular context.”

The absence of a thermal stability difference cannot be taken as evidence that the protein is conformationally unperturbed.

“the absence of a thermal stability difference cannot be taken as evidence that the protein is conformationally unperturbed”

Mutations in certain regions of ApoE are more likely to impact thermal stability than others.

“Our findings reveal that mutations in certain regions of ApoE are more likely to impact thermal stability than others.”

The concordance between brain tissue CETSA and cellular BiTSA results validates the use of BiTSA for systematic characterization of ApoE variants.

“The concordance between brain tissue CETSA and cellular BiTSA results validates the use of BiTSA for systematic characterization of ApoE variants.”

Differences in absolute Tm values between contexts almost certainly indicate the presence of different ApoE binding partners or lipids.

“Notably, while the delta between ApoE3 and ApoE4 was conserved across contexts, there were differences in absolute values between contexts, which almost certainly indicates the presence of different ApoE binding partners or lipids.”

The consistency of our findings across different experimental systems (CETSA in brain tissue and BiTSA in cultured cells) supports the validity of our conclusions.

“Despite these limitations, the consistency of our findings across different experimental systems (CETSA in brain tissue and BiTSA in cultured cells) supports the validity of our conclusions.”

This approach enabled us to examine a larger panel of variants than would be feasible with tissue-based methods alone.

“This approach enabled us to examine a larger panel of variants than would be feasible with tissue-based methods alone.”

These complex relationships have implications for understanding the structural basis of ApoE-associated disease risk.

“Our findings reveal complex relationships between ApoE sequence, isoform background, and thermal stability that have implications for understanding the structural basis of ApoE-associated disease risk.”