Clin Exp Nephrol DOI 10.1007/s10157-013-0886-5
REVIEW ARTICLE
WCN 2013 Satellite Symposium ‘‘Kidney and Lipids’’
A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy Efstratios Stratikos • Angeliki Chroni
Received: 28 August 2013 / Accepted: 1 October 2013 Ó Japanese Society of Nephrology 2013
Abstract Single amino acid mutations in apolipoprotein E (apoE) have been associated with the development of the rare kidney disease lipoprotein glomerulopathy (LPG). Although the genetic linkage to disease development is well established, the mechanism of pathogenesis is largely unknown, limiting therapeutic insight. Here, we summarize current knowledge in the field and focus on the possible effects of LPG-associated mutations on the structure of apoE. Recent findings have suggested that mutationinduced folding perturbations in apoE lead to structural destabilization and aggregation, effects that may underlie lipoprotein thrombi accumulation in the glomerulus, a hallmark of LPG. The recognition that structural destabilization may underlie the association between apoE mutations and LPG can be key for development of new innovative treatments for this rare disease. Keywords Apolipoprotein E Lipoprotein glomerulopathy Lipoproteins Structure Biophysics Mutation Destabilization Aggregation
glomerular capillaries. After its initial discovery, it appeared that the disease was restricted to individuals from East Asia but during recent years several cases have been reported in Europe and the Americas [1, 3–6]. Renal transplantation does not cure the disease and invariably leads to disease recurrence suggesting that a factor outside the kidney is key to the pathogenesis [7]. Indeed, LPG has been shown to have a strong genetic predisposition component with mutations within the apolipoprotein E gene (apoE) acting in a dominant manner but with incomplete penetrance [8]. The importance of apoE in the pathogenesis of the disease has been confirmed by gene transfer studies of the naturally occurring apoE variant, apoE Sendai (R145P) to apoE-deficient mice that leads to lipoprotein depositions in the glomerulus of the kidney [9]. These findings have suggested that apoE dysfunction may be an etiological cause of LPG, although the exact mechanism remains elusive.
Structure of apoE: plasticity, conformational changes and thermodynamic instability Apolipoprotein E rare mutations and lipoprotein glomerulopathy Lipoprotein glomerulopathy (LPG) is a rare disease of the kidney that can lead to kidney failure [1, 2]. It is characterized by the formation of lipoprotein thrombi in the E. Stratikos (&) A. Chroni National Center for Scientific Research Demokritos, Patriarhou Gregoriou and Neapoleos Street, 15310 Agia Paraskevi, Greece e-mail: [email protected]; [email protected] A. Chroni e-mail: [email protected]
ApoE is a major protein component of the lipoprotein transport system in humans and plays critical roles in the pathogenesis of atherosclerosis and Alzheimer’s disease [10]. It consists of 299 residues and has three common isoforms in humans (apoE2, apoE3 and apoE4) differing at positions 112 and 158 [11]. ApoE can interact with lipids and is a functional and structural component of lipoprotein particles such as chylomicrons, chylomicron remnants and very low-density lipoprotein, as well as intermediate-density lipoprotein and high-density lipoprotein. In its lipidfree form it displays unprecedented structural flexibility, a property linked with its ability to function correctly [12].
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ApoE presents two different functional domains that can be separated after thrombin digestion—a 22 kDa N-terminal domain and a smaller 10 kDa C-terminal domain [13, 14]. The N-terminal domain has been analyzed by X-ray crystallography and forms a four-helix bundle. The C-terminal domain is also highly helical but appears to be more flexible and to facilitate unfolding of the N-terminal domain during the process of lipid binding. Recently, a full-length structure of an artificial variant of apoE3 has been released, determined by nuclear magnetic resonance [15]. This variant contains 5 amino acid substitutions that stabilize the protein in a monomeric form to allow structure determination. In this structure, that almost certainly corresponds to a snapshot of the possible conformations of apoE, the N-terminal domain retains the four-helix bundle configuration, but the C-terminal domain folds over and around the N-terminal domain, covering the LDL receptor binding region of apoE (amino acids 136–150). ApoE has a much lower thermodynamic stability compared to other globular proteins, a property linked with its ability to readily change conformations. Its reduced stability arises in part due to the interaction of the N-terminal with the C-terminal domain [16]. Several mutations in the gene of apoE have been linked with predisposition to disease and later shown to affect the thermodynamic stability of the protein, including the natural polymorphisms defining the apoE2, apoE3 and apoE4 isoforms [17–23].
Location of LPG-associated mutations Several single amino acid mutations within apoE have been described in the literature to be associated with the development of LPG (Table 1). Almost all of them are located in the N-terminal domain of the molecule, with the majority located on helix 4 of the four-helix bundle and corresponding to Arginine (Arg) to Proline (Pro) or Cysteine (Cys) substitutions (Fig. 1). Mutation of residues at or near the LDL-receptor family binding region of apoE may affect apoE clearance and normal lipid metabolism although it is not clear how such effects lead to LPG pathogenesis. ApoE Sendai has a 20-fold reduced LDL receptor binding capacity but near normal heparan sulfate proteoglycan binding, suggesting impaired apoE clearance from the glomerulus [24]. ApoE Kyoto also has reduced LDL receptor family binding capacity and triglyceride-rich lipoproteins containing apoE Kyoto have 30–50 % increased binding to human umbilical vein endothelial cells [25, 26]. ApoE Chicago also demonstrated enhanced binding to the wall of the glomerular capillary [4]. These results suggest that altered binding to and/or clearance from endothelial cell surface of the mutant apoE proteins may contribute to deposition although little is known on
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Table 1 Apolipoprotein E variants associated with the development of LPG Variant name
Amino acid substitution
Reference
apoE Kyoto
apoE3 (Arg25Cys)
[25]
apoE Tsukuba
apoE3 (Arg114Cys)
[33]
apoE Tokyo
apoE3 [del(Leu141Lys143)] apoE3 [del(Arg142Leu144)]
[34]
apoE Maebashi
[35, 36]
apoE Sendai
apoE3 (Arg145Pro)
[37]
apoE Chicago
apoE3 (Arg147Pro)
[4]
apoE Guangzhou
apoE3 (Arg150Pro)
[38]
apoE Okayama
apoE2 [Arg150Gly)
[39]
apoE Modena
apoE2 (Arg150Cys)
[6]
apoE Las Vegas
apoE3 (Ala152Asp)
[5]
apoE Osaka or Kurashiki
apoE3 (Arg158Pro)
[40, 41]
apoE1 [del(Gln156Lys173)]
apoE3 [del(Gln156Lys173)]
[42]
apoE Hong Kong
apoE3 (Asp230Tyr)
[43]
Arg114Cys (Tsukuba) Arg147Pro (Chicago)
Arg150Cys (Modena)
Arg145Pro (Sendai)
Ala152Asp (Las Vegas) Arg25Cys (Kyoto)
Arg158Pro (Osaka)
Fig. 1 Schematic representation of LPG-associated mutations in apoE. The N-terminal domain of apoE is represented in cartoon format and the mutated residues in sphere format. Figure generated using coordinates from the crystal structure of apoE3 with pdb code 1LPE
why this would be exclusive to the glomerulus. Furthermore, the common variant apoE2 binds the LDL receptor 50-fold weaker than apoE3, but evidence demonstrating a clear association between apoE2 and LPG is very limited since the renal lipidosis occasionally observed in apoE2
Clin Exp Nephrol
homozygotes suffering from type III hyperlipoproteinemia lacks lipoprotein thrombi [27]. Overall, although LDL receptor-mediated clearance may be a contributing factor to LPG pathogenesis, other factors must also play important roles. An alternative concept for understanding the role of the apoE mutations to the pathogenesis of LPG can arise from examining the potential structural repercussions of the LPG-associated mutations and in particular the nature of the amino acid substitution and its expected effect on structure and folding of the protein. Both proline and cysteine are amino acids that have unique properties that can be of the upmost importance for protein structure. Proline, the only natural imino acid is structurally rigid and is often incompatible with helical segments. On the other hand, cysteine can form disulfide bonds either with another cysteine within the same molecule or with a different molecule. As a result, both types of substitutions can in theory lead to structural perturbations in apoE.
Possible effects on structure: thermodynamic and structural findings Following the rational described above, we decided to examine the structural ramifications of single amino acid substitutions in apoE that have been associated with LPG pathogenesis. We initially examined the effects brought about to apoE3 by three Arg to Pro substitutions at positions 145, 147 and 158 [28]. Using an array of biophysical techniques, we discovered that apoE3 variants carrying these mutations displayed major thermodynamic destabilization, structural perturbations, partial hydrophobic core exposure and were aggregation prone. Interestingly, a different set of mutations that reside in the same general area of apoE3, but are instead associated with the development of type III hyperlipoproteinemia, were found to induce only minor thermodynamic perturbations suggesting that the nature of the introduced amino acid is key to its effect of structure [18]. Our findings, in combination with previous knowledge about the structure and folding of apoE3, suggested that the N-terminal domain of the apoE protein, containing Arg to Pro substitutions at positions 145, 147 and 158, was deficient in folding, leading to partially unfolded states in the mature protein. These phenomena translated to lipidated states of the protein which were also prone to aggregation and presented structurally aberrant sub-populations [28]. The findings summarized in the paragraph above are generally consistent with the incompatibility of proline in the highly helical N-terminal segment of apoE3. The same pattern may not be, at first glance, expected for cysteine substitutions since cysteine is often found in helical
segments of protein structures. Preliminary analysis, however, suggests that cysteine substitutions associated with LPG can result to equally significant perturbations in apoE structure (E. Stratikos and A. Chroni, unpublished results). It remains to be determined whether this phenomenon is related to the ability of cysteine to form disulfide bonds within the molecule of apoE3 during folding or other structural transitions.
Unfolding and aggregation as a pathogenic mechanism A large array of human diseases is considered to be initiated due to protein aggregation that eventually leads to cellular or organ toxicity. Such examples include Alzheimer’s disease, prion disease and many types of amyloidosis. The general motif for the pathogenesis of such diseases includes the generation of unstable protein molecules either due to mutation, proteolysis or induction of folding defects leading to aggregation and formation of insoluble particles that interfere with normal cellular or organ function. Although it is not clear whether LPG is initiated due to a similar pathogenic mechanism, the finding that LPGassociated apoE mutations lead to folding defects and protein or lipoprotein aggregation, suggests that this may be at least in part a component of the pathogenic mechanism. The accumulated thrombi in the kidneys of LPG patients however are primarily of lipoprotein nature, suggesting that the structural defects found in lipoprotein particles containing the mutant apoEs may be more relevant compared to the defects on the lipid-free protein. Furthermore, the sensitivity of lipoprotein particles containing mutant apoE3 variants to secretory phospholipase A2 may provide another pathway to lipoprotein aggregation [18]. In either case, the increased mechanical pressure native to the glomerulus during the normal filtration process may provide a unique microenvironment that promotes protein aggregation either by generating distinct composition compared to normal sera or by just leading to increased local concentration of lipoproteins, enhancing aggregation kinetics.
Insights into possible therapies: structure correctors If structural and folding perturbations leading to protein aggregation are indeed major factors behind the role of apoE in the pathogenesis of LPG then the pharmacological manipulation of apoE structure may be a promising approach for the treatment of LPG. An example of such an approach has been recently described for apoE4 [29]. This apoE allele is known to be associated with increased risk for development of late-onset Alzheimer’s disease and
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although it only differs from the more common apoE3 allele in a single amino acid, structural and thermodynamic differences have been established and hypothesized to underlie the different pathogenic role of the two alleles [30]. In a recent series of studies the authors have demonstrated small-molecular weight compounds able to affect apoE4 folding so that the final molecule is structurally closer to apoE3 [31, 32]. These compounds can reduce the neurotoxic effects of apoE4, as well as the proteolytic susceptibility in vivo of apoE4. Such ‘structure correctors’ are hypothesized to influence folding pathways and stabilize the correct final structure that has been perturbed by mutation. It may be possible to develop similar functioning compounds that correct the N-terminal misfolding of the variants that are associated with LPG leading to stabilized apoE, in both the lipid-free and lipidated form and stopping aggregation. Such an approach may hold promise in controlling or even reversing lipoprotein thrombi deposition in the kidney, something that holds obvious therapeutic value. However, the discovery of such compounds for apoE4 requires detailed knowledge on the structural differences between apoE3 and apoE4. As a result more work is necessary to help us understand the exact structural perturbations brought about by LPG-associated apoE mutations before similar efforts can be successful. Conflict of interest interest exists.
The authors have declared that no conflict of
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9. Ishigaki Y, Oikawa S, Suzuki T, Usui S, Magoori K, Kim DH, et al. Virus-mediated transduction of apolipoprotein E (ApoE)Sendai develops lipoprotein glomerulopathy in ApoE-deficient mice. J Biol Chem. 2000;275(40):31269–73. 10. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 1988;240(4852):622–30. 11. Zannis VI, Breslow JL, Utermann G, Mahley RW, Weisgraber KH, Havel RJ, et al. Proposed nomenclature of apoE isoproteins, apoE genotypes, and phenotypes. J Lipid Res. 1982;23(6):911–4. 12. Mahley RW, Weisgraber KH, Huang Y. Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer’s disease to AIDS. J Lipid Res. 2009;50 Suppl:S183–8. 13. Aggerbeck LP, Wetterau JR, Weisgraber KH, Wu CS, Lindgren FT. Human apolipoprotein E3 in aqueous solution II. Properties of the amino- and carboxyl-terminal domains. J Biol Chem. 1988;263(13):6249–58. 14. Wetterau JR, Aggerbeck LP, Rall SC Jr, Weisgraber KH. Human apolipoprotein E3 in aqueous solution I. Evidence for two structural domains. J Biol Chem. 1988;263(13):6240–8. 15. Chen J, Li Q, Wang J. Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions. Proc Natl Acad Sci USA. 2011;108(36):14813–8. 16. Chroni A, Pyrpassopoulos S, Thanassoulas A, Nounesis G, Zannis VI, Stratikos E. Biophysical analysis of progressive C-terminal truncations of human apolipoprotein E4: insights into secondary structure and unfolding properties. Biochemistry. 2008;47(35):9071–80. 17. Mahley RW, Rall SC Jr. Type III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2001. 18. Georgiadou D, Chroni A, Vezeridis A, Zannis VI, Stratikos E. Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia. PLoS One. 2011;6(11):e27037. 19. Nguyen D, Dhanasekaran P, Nickel M, Nakatani R, Saito H, Phillips MC, et al. Molecular basis for the differences in lipid and lipoprotein binding properties of human apolipoproteins E3 and E4. Biochemistry. 2010;49(51):10881–9. 20. Johnson LA, Arbones-Mainar JM, Fox RG, Pendse AA, Altenburg MK, Kim HS, et al. Apolipoprotein E4 exaggerates diabetic dyslipidemia and atherosclerosis in mice lacking the LDL receptor. Diabetes. 2011;60(9):2285–94. 21. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science. 1993;261(5123):921–3. 22. Dong LM, Weisgraber KH. Human apolipoprotein E4 domain interaction. Arginine 61 and glutamic acid 255 interact to direct the preference for very low density lipoproteins. J Biol Chem. 1996;271(32):19053–7. 23. Dong LM, Parkin S, Trakhanov SD, Rupp B, Simmons T, Arnold KS, et al. Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia. Nat Struct Biol. 1996;3(8):718–22. 24. Hoffmann M, Scharnagl H, Panagiotou E, Banghard W, Wieland H, Marz W. Diminished LDL receptor and high heparin binding of apolipoprotein E2 Sendai associated with lipoprotein glomerulopathy. J Am Soc Nephrol. 2001;12(3):524–30. 25. Matsunaga A, Sasaki J, Komatsu T, Kanatsu K, Tsuji E, Moriyama K, et al. A novel apolipoprotein E mutation, E2 (Arg25Cys), in lipoprotein glomerulopathy. Kidney Int. 1999;56(2): 421–7. 26. Murano T, Matsumura R, Misawa Y, Ozaki H, Miyashita Y, Yoshida S, et al. Interaction of endothelial cells and triglyceriderich lipoproteins with apolipoprotein E (Arg ? Cys) from a
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35. Ogawa T, Maruyama K, Hattori H, Arai H, Kondoh I, Egashira T, et al. A new variant of apolipoprotein E (apo E Maebashi) in lipoprotein glomerulopathy. Pediatr Nephrol. 2000;14(2):149–51. 36. Maruyama K, Arai H, Ogawa T, Tomizawa S, Morikawa A. Lipoprotein glomerulopathy: a pediatric case report. Pediatr Nephrol. 1997;11(2):213–4. 37. Oikawa S, Matsunaga A, Saito T, Sato H, Seki T, Hoshi K, et al. Apolipoprotein E Sendai (arginine 145 ? proline): a new variant associated with lipoprotein glomerulopathy. J Am Soc Nephrol. 1997;8(5):820–3. 38. Luo B, Huang F, Liu Q, Li X, Chen W, Zhou SF, et al. Identification of apolipoprotein E Guangzhou (arginine 150 proline), a new variant associated with lipoprotein glomerulopathy. Am J Nephrol. 2008;28(2):347–53. 39. Kinomura M, Sugiyama H, Saito T, Matsunaga A, Sada KE, Kanzaki M, et al. A novel variant apolipoprotein E Okayama in a patient with lipoprotein glomerulopathy. Nephrol Dial Transplant. 2008;23(2):751–6. 40. Mitani A, Ishigami M, Watase K, Minakata T, Yamamura T. A novel apolipoprotein E mutation, ApoE Osaka (Arg158 Pro), in a dyslipidemic patient with lipoprotein glomerulopathy. J Atheroscler Thromb. 2011;18(6):531–5. 41. Tokura T, Itano S, Kobayashi S, Kuwabara A, Fujimoto S, Horike H, et al. A novel mutation ApoE2 Kurashiki (R158P) in a patient with lipoprotein glomerulopathy. J Atheroscler Thromb. 2011;18(6):536–41. 42. Ando M, Sasaki J, Hua H, Matsunaga A, Uchida K, Jou K, et al. A novel 18-amino acid deletion in apolipoprotein E associated with lipoprotein glomerulopathy. Kidney Int. 1999;56(4): 1317–23. 43. Cheung CY, Chan AO, Chan YH, Lee KC, Chan GP, Lau GT, et al. A rare cause of nephrotic syndrome: lipoprotein glomerulopathy. Hong Kong Med J. 2009;15(1):57–60.
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REVIEW ARTICLE
WCN 2013 Satellite Symposium ‘‘Kidney and Lipids’’
A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy Efstratios Stratikos • Angeliki Chroni
Received: 28 August 2013 / Accepted: 1 October 2013 Ó Japanese Society of Nephrology 2013
Abstract Single amino acid mutations in apolipoprotein E (apoE) have been associated with the development of the rare kidney disease lipoprotein glomerulopathy (LPG). Although the genetic linkage to disease development is well established, the mechanism of pathogenesis is largely unknown, limiting therapeutic insight. Here, we summarize current knowledge in the field and focus on the possible effects of LPG-associated mutations on the structure of apoE. Recent findings have suggested that mutationinduced folding perturbations in apoE lead to structural destabilization and aggregation, effects that may underlie lipoprotein thrombi accumulation in the glomerulus, a hallmark of LPG. The recognition that structural destabilization may underlie the association between apoE mutations and LPG can be key for development of new innovative treatments for this rare disease. Keywords Apolipoprotein E Lipoprotein glomerulopathy Lipoproteins Structure Biophysics Mutation Destabilization Aggregation
glomerular capillaries. After its initial discovery, it appeared that the disease was restricted to individuals from East Asia but during recent years several cases have been reported in Europe and the Americas [1, 3–6]. Renal transplantation does not cure the disease and invariably leads to disease recurrence suggesting that a factor outside the kidney is key to the pathogenesis [7]. Indeed, LPG has been shown to have a strong genetic predisposition component with mutations within the apolipoprotein E gene (apoE) acting in a dominant manner but with incomplete penetrance [8]. The importance of apoE in the pathogenesis of the disease has been confirmed by gene transfer studies of the naturally occurring apoE variant, apoE Sendai (R145P) to apoE-deficient mice that leads to lipoprotein depositions in the glomerulus of the kidney [9]. These findings have suggested that apoE dysfunction may be an etiological cause of LPG, although the exact mechanism remains elusive.
Structure of apoE: plasticity, conformational changes and thermodynamic instability Apolipoprotein E rare mutations and lipoprotein glomerulopathy Lipoprotein glomerulopathy (LPG) is a rare disease of the kidney that can lead to kidney failure [1, 2]. It is characterized by the formation of lipoprotein thrombi in the E. Stratikos (&) A. Chroni National Center for Scientific Research Demokritos, Patriarhou Gregoriou and Neapoleos Street, 15310 Agia Paraskevi, Greece e-mail: [email protected]; [email protected] A. Chroni e-mail: [email protected]
ApoE is a major protein component of the lipoprotein transport system in humans and plays critical roles in the pathogenesis of atherosclerosis and Alzheimer’s disease [10]. It consists of 299 residues and has three common isoforms in humans (apoE2, apoE3 and apoE4) differing at positions 112 and 158 [11]. ApoE can interact with lipids and is a functional and structural component of lipoprotein particles such as chylomicrons, chylomicron remnants and very low-density lipoprotein, as well as intermediate-density lipoprotein and high-density lipoprotein. In its lipidfree form it displays unprecedented structural flexibility, a property linked with its ability to function correctly [12].
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ApoE presents two different functional domains that can be separated after thrombin digestion—a 22 kDa N-terminal domain and a smaller 10 kDa C-terminal domain [13, 14]. The N-terminal domain has been analyzed by X-ray crystallography and forms a four-helix bundle. The C-terminal domain is also highly helical but appears to be more flexible and to facilitate unfolding of the N-terminal domain during the process of lipid binding. Recently, a full-length structure of an artificial variant of apoE3 has been released, determined by nuclear magnetic resonance [15]. This variant contains 5 amino acid substitutions that stabilize the protein in a monomeric form to allow structure determination. In this structure, that almost certainly corresponds to a snapshot of the possible conformations of apoE, the N-terminal domain retains the four-helix bundle configuration, but the C-terminal domain folds over and around the N-terminal domain, covering the LDL receptor binding region of apoE (amino acids 136–150). ApoE has a much lower thermodynamic stability compared to other globular proteins, a property linked with its ability to readily change conformations. Its reduced stability arises in part due to the interaction of the N-terminal with the C-terminal domain [16]. Several mutations in the gene of apoE have been linked with predisposition to disease and later shown to affect the thermodynamic stability of the protein, including the natural polymorphisms defining the apoE2, apoE3 and apoE4 isoforms [17–23].
Location of LPG-associated mutations Several single amino acid mutations within apoE have been described in the literature to be associated with the development of LPG (Table 1). Almost all of them are located in the N-terminal domain of the molecule, with the majority located on helix 4 of the four-helix bundle and corresponding to Arginine (Arg) to Proline (Pro) or Cysteine (Cys) substitutions (Fig. 1). Mutation of residues at or near the LDL-receptor family binding region of apoE may affect apoE clearance and normal lipid metabolism although it is not clear how such effects lead to LPG pathogenesis. ApoE Sendai has a 20-fold reduced LDL receptor binding capacity but near normal heparan sulfate proteoglycan binding, suggesting impaired apoE clearance from the glomerulus [24]. ApoE Kyoto also has reduced LDL receptor family binding capacity and triglyceride-rich lipoproteins containing apoE Kyoto have 30–50 % increased binding to human umbilical vein endothelial cells [25, 26]. ApoE Chicago also demonstrated enhanced binding to the wall of the glomerular capillary [4]. These results suggest that altered binding to and/or clearance from endothelial cell surface of the mutant apoE proteins may contribute to deposition although little is known on
123
Table 1 Apolipoprotein E variants associated with the development of LPG Variant name
Amino acid substitution
Reference
apoE Kyoto
apoE3 (Arg25Cys)
[25]
apoE Tsukuba
apoE3 (Arg114Cys)
[33]
apoE Tokyo
apoE3 [del(Leu141Lys143)] apoE3 [del(Arg142Leu144)]
[34]
apoE Maebashi
[35, 36]
apoE Sendai
apoE3 (Arg145Pro)
[37]
apoE Chicago
apoE3 (Arg147Pro)
[4]
apoE Guangzhou
apoE3 (Arg150Pro)
[38]
apoE Okayama
apoE2 [Arg150Gly)
[39]
apoE Modena
apoE2 (Arg150Cys)
[6]
apoE Las Vegas
apoE3 (Ala152Asp)
[5]
apoE Osaka or Kurashiki
apoE3 (Arg158Pro)
[40, 41]
apoE1 [del(Gln156Lys173)]
apoE3 [del(Gln156Lys173)]
[42]
apoE Hong Kong
apoE3 (Asp230Tyr)
[43]
Arg114Cys (Tsukuba) Arg147Pro (Chicago)
Arg150Cys (Modena)
Arg145Pro (Sendai)
Ala152Asp (Las Vegas) Arg25Cys (Kyoto)
Arg158Pro (Osaka)
Fig. 1 Schematic representation of LPG-associated mutations in apoE. The N-terminal domain of apoE is represented in cartoon format and the mutated residues in sphere format. Figure generated using coordinates from the crystal structure of apoE3 with pdb code 1LPE
why this would be exclusive to the glomerulus. Furthermore, the common variant apoE2 binds the LDL receptor 50-fold weaker than apoE3, but evidence demonstrating a clear association between apoE2 and LPG is very limited since the renal lipidosis occasionally observed in apoE2
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homozygotes suffering from type III hyperlipoproteinemia lacks lipoprotein thrombi [27]. Overall, although LDL receptor-mediated clearance may be a contributing factor to LPG pathogenesis, other factors must also play important roles. An alternative concept for understanding the role of the apoE mutations to the pathogenesis of LPG can arise from examining the potential structural repercussions of the LPG-associated mutations and in particular the nature of the amino acid substitution and its expected effect on structure and folding of the protein. Both proline and cysteine are amino acids that have unique properties that can be of the upmost importance for protein structure. Proline, the only natural imino acid is structurally rigid and is often incompatible with helical segments. On the other hand, cysteine can form disulfide bonds either with another cysteine within the same molecule or with a different molecule. As a result, both types of substitutions can in theory lead to structural perturbations in apoE.
Possible effects on structure: thermodynamic and structural findings Following the rational described above, we decided to examine the structural ramifications of single amino acid substitutions in apoE that have been associated with LPG pathogenesis. We initially examined the effects brought about to apoE3 by three Arg to Pro substitutions at positions 145, 147 and 158 [28]. Using an array of biophysical techniques, we discovered that apoE3 variants carrying these mutations displayed major thermodynamic destabilization, structural perturbations, partial hydrophobic core exposure and were aggregation prone. Interestingly, a different set of mutations that reside in the same general area of apoE3, but are instead associated with the development of type III hyperlipoproteinemia, were found to induce only minor thermodynamic perturbations suggesting that the nature of the introduced amino acid is key to its effect of structure [18]. Our findings, in combination with previous knowledge about the structure and folding of apoE3, suggested that the N-terminal domain of the apoE protein, containing Arg to Pro substitutions at positions 145, 147 and 158, was deficient in folding, leading to partially unfolded states in the mature protein. These phenomena translated to lipidated states of the protein which were also prone to aggregation and presented structurally aberrant sub-populations [28]. The findings summarized in the paragraph above are generally consistent with the incompatibility of proline in the highly helical N-terminal segment of apoE3. The same pattern may not be, at first glance, expected for cysteine substitutions since cysteine is often found in helical
segments of protein structures. Preliminary analysis, however, suggests that cysteine substitutions associated with LPG can result to equally significant perturbations in apoE structure (E. Stratikos and A. Chroni, unpublished results). It remains to be determined whether this phenomenon is related to the ability of cysteine to form disulfide bonds within the molecule of apoE3 during folding or other structural transitions.
Unfolding and aggregation as a pathogenic mechanism A large array of human diseases is considered to be initiated due to protein aggregation that eventually leads to cellular or organ toxicity. Such examples include Alzheimer’s disease, prion disease and many types of amyloidosis. The general motif for the pathogenesis of such diseases includes the generation of unstable protein molecules either due to mutation, proteolysis or induction of folding defects leading to aggregation and formation of insoluble particles that interfere with normal cellular or organ function. Although it is not clear whether LPG is initiated due to a similar pathogenic mechanism, the finding that LPGassociated apoE mutations lead to folding defects and protein or lipoprotein aggregation, suggests that this may be at least in part a component of the pathogenic mechanism. The accumulated thrombi in the kidneys of LPG patients however are primarily of lipoprotein nature, suggesting that the structural defects found in lipoprotein particles containing the mutant apoEs may be more relevant compared to the defects on the lipid-free protein. Furthermore, the sensitivity of lipoprotein particles containing mutant apoE3 variants to secretory phospholipase A2 may provide another pathway to lipoprotein aggregation [18]. In either case, the increased mechanical pressure native to the glomerulus during the normal filtration process may provide a unique microenvironment that promotes protein aggregation either by generating distinct composition compared to normal sera or by just leading to increased local concentration of lipoproteins, enhancing aggregation kinetics.
Insights into possible therapies: structure correctors If structural and folding perturbations leading to protein aggregation are indeed major factors behind the role of apoE in the pathogenesis of LPG then the pharmacological manipulation of apoE structure may be a promising approach for the treatment of LPG. An example of such an approach has been recently described for apoE4 [29]. This apoE allele is known to be associated with increased risk for development of late-onset Alzheimer’s disease and
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although it only differs from the more common apoE3 allele in a single amino acid, structural and thermodynamic differences have been established and hypothesized to underlie the different pathogenic role of the two alleles [30]. In a recent series of studies the authors have demonstrated small-molecular weight compounds able to affect apoE4 folding so that the final molecule is structurally closer to apoE3 [31, 32]. These compounds can reduce the neurotoxic effects of apoE4, as well as the proteolytic susceptibility in vivo of apoE4. Such ‘structure correctors’ are hypothesized to influence folding pathways and stabilize the correct final structure that has been perturbed by mutation. It may be possible to develop similar functioning compounds that correct the N-terminal misfolding of the variants that are associated with LPG leading to stabilized apoE, in both the lipid-free and lipidated form and stopping aggregation. Such an approach may hold promise in controlling or even reversing lipoprotein thrombi deposition in the kidney, something that holds obvious therapeutic value. However, the discovery of such compounds for apoE4 requires detailed knowledge on the structural differences between apoE3 and apoE4. As a result more work is necessary to help us understand the exact structural perturbations brought about by LPG-associated apoE mutations before similar efforts can be successful. Conflict of interest interest exists.
The authors have declared that no conflict of
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