International Journal of Molecular Sciences Communication Differences in Recycling of Apolipoprotein E3 and E4—LDL Receptor Complexes—A Mechanistic Hypothesis Meewhi Kim 1,* and Ilya Bezprozvanny 1,2,* 1 Department of Physiology, UT Southwestern Medical Center, Dallas, TX 75390, USA 2 Laboratory of Molecular Neurodegeneration, Peter the Great St. Petersburg State Polytechnic University, 195251 St. Petersburg, Russia * Correspondence: [email protected] (M.K.); [email protected] (I.B.) Abstract: Apolipoprotein E (ApoE) is a protein that plays an important role in the transport of fatty acids and cholesterol and in cellular signaling. On the surface of the cells, ApoE lipoparticles bind to low density lipoprotein receptors (LDLR) that mediate the uptake of the lipids and downstream signaling events. There are three alleles of the human ApoE gene. Presence of ApoE4 allele is a major risk factor for developing Alzheimer’s disease (AD) and other disorders late in life, but the mechanisms responsible for biological differences between different ApoE isoforms are not well understood. We here propose that the differences between ApoE isoforms can be explained by differences in the pH-dependence of the association between ApoE3 and ApoE4 isoforms and LDL-A repeats of LDLR. As a result, the following endocytosis ApoE3-associated LDLRs are recycled back to the plasma membrane but ApoE4-containing LDLR complexes are trapped in late endosomes and targeted for degradation. The proposed mechanism is predicted to lead to a reduction in steady- state surface levels of LDLRs and impaired cellular signaling in ApoE4-expressing cells. We hope that this proposal will stimulate experimental research in this direction that allows the testing of Citation: Kim, M.; Bezprozvanny, I. our hypothesis. Differences in Recycling of Apolipoprotein E3 and E4—LDL Keywords: ApoE; LDL receptor; endosome; Alzheimer’s disease; modelling; protonation; Receptor Complexes—A Mechanistic charged interaction Hypothesis. Int. J. Mol. Sci. 2021, 22, 5030. https://doi.org/10.3390/ ijms22095030 1. Introduction Academic Editor: Michael Ugrumov Apolipoprotein E (ApoE) is a protein that plays an important role in the transport Received: 26 April 2021 of fatty acids and cholesterol and in cellular signaling [1–4]. On the surface of the cells, Accepted: 7 May 2021 ApoE lipoparticles bind to Low Density Lipoprotein receptors (LDLR) that mediate the Published: 10 May 2021 uptake of the lipids and downstream signaling events [1–4]. There are three alleles of the human ApoE gene—E2, E3, and E4—with two of these alleles co-expressed in many tissues, Publisher’s Note: MDPI stays neutral including the central nervous system [4–8]. These alleles differ in amino acids in positions with regard to jurisdictional claims in 112 and 158 of N-terminal ligand domains as follows—E2 (C112, C158), E3(C112, R158) published maps and institutional affil- and E4(R112, R158). There is well established genetic association between the presence of iations. different alleles of the ApoE gene and human diseases [9–13]. In particular, the presence of ApoE4 is a major risk factor for developing Alzheimer’s disease (AD) late in life [14–20]. The mechanisms responsible for biological differences between different ApoE isoforms are not well understood and are currently under intense investigation [21–23]. Copyright: © 2021 by the authors. The ApoE protein is composed of the amino-terminal (N) and carboxy-terminal (C) Licensee MDPI, Basel, Switzerland. domains, which are loosely hinged to each other [24–26]. The N-terminal ligand domain This article is an open access article (LD) is involved in the association with surface receptors including LDLR (Figure1A, distributed under the terms and Supplementary Figure S1); the C-terminal domain is involved in the binding of cholesterol conditions of the Creative Commons and fatty acids [27–29]. Both domains form α-helical bundles [24] with the critical allelic Attribution (CC BY) license (https:// positions C112 and C158, which are located on the side of the fourth and third α-helixes in creativecommons.org/licenses/by/ the ApoE-LD domain. 4.0/). Int. J. Mol. Sci. 2021, 22, 5030. https://doi.org/10.3390/ijms22095030 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x Int. J. Mol. Sci. 2021, 22, 5030 2 of 10 2 of 10 Figure 1. Ligand domain (LD) of ApoE and LDL-A repeats of LDLR. (A) The secondary structure of ApoE-LD and the domainFigure structure 1. Ligand of LDLR domain are shown. (LD) The of receptor-bindingApoE and LDL interface‐A repeats is a partof LDLR. of the fourth(A) Theα-helix secondary in the ApoE-LD structure domain (shownof ApoE in blue).‐LD The and R112C the domain domain portionstructure of theof LDLR third α are-helix shown. in the The ApoE-LD receptor domain‐binding is shown interface in orange. is a part Position 112 withinof the the fourth R112C α‐ domainhelix in is the shown ApoE in purple.‐LD domain Seven amino-terminal(shown in blue). LDL-A The repeatsR112C aredomain shown portion as colored of semicircles.the (B) Primarythird α‐ sequencehelix in alignment the ApoE of‐LD R112C domain domains is shown from ApoE4 in orange. and ApoE3 Position isoforms. 112 Aminowithin acid the inR112C position domain 112 is shownis in redshown (R for ApoE4 in purple. and CSeven for ApoE3). amino (‐Cterminal) Primary LDL sequences‐A repeats of seven are LDL-Ashown repeats as colored from semicircles. the LDLR. The (B sequences) Pri‐ of RP1–RP7mary repeats sequence are color-coded alignment according of R112C to domains the panel Afrom diagram. ApoE4 and ApoE3 isoforms. Amino acid in position 112 is shown in red (R for ApoE4 and C for ApoE3). (C) Primary sequences of seven LDL‐ A repeats from the LDLR.LDLR The sequences is a single transmembraneof RP1–RP7 repeats domain are color protein‐coded that containsaccording seven to the class A repeats panel A diagram. (LDL-A) on its amino terminal (Figure1A, Supplementary Figure S1) [ 30]. These repeats are approximately 40 amino acids long and constrained by three internal disulphide bridges LDLR is a singlethat transmembrane contain conserved domain DCXDXSDE protein motifs that [31 contains] (Supplementary seven class Figure A repeats S1B). These do- (LDL‐A) on its aminomains terminal are involved (Figure in the 1A, association Supplementary with extracellular Figure S1) ligands [30]. [ 2These,6,32]. Therepeats cytosolic tail of LDLR mediates signal transduction and endocytosis [5,33–35]. Similar to other LDLR are approximatelyligands, 40 amino ApoE acids lipoparticles long and bind constrained to LDL-A amino-terminal by three internal repeats disulphide [36–41] (Figure 1A , bridges that containSupplementary conserved Figure DCXDXSDE S1). A ligand motifs binding [31] to (Supplementary the LDLR triggers cellularFigure signalingS1B). trans- These domains areduction involved and in the the internalization association ofwith the extracellular ligand–receptor ligands complex [2,6,32]. via clathrin-assisted The cy‐ en- tosolic tail of LDLRdocytosis mediates [1]. Thesignal internalized transduction complex and is sortedendocytosis to endosomal [5,33–35]. compartments Similar to where the other LDLR ligands,ligand ApoE is released lipoparticles and LDLR bind is recycled to LDL to‐A the amino plasma‐terminal membrane repeats [42–44]. [36–41] Multiple sources (Figure 1A, Supplementaryof evidence Figure demonstrate S1). A that ligand LDLR binding recycling to the requires LDLR organized triggers cellular cellular actions sig‐ includ- ing luminal endosomal acidification and calcium (Ca2+) signaling [45–51]. In particular, naling transduction and the internalization of the ligand–receptor complex via clathrin‐ endosomal acidification plays a key role in the control of LDLR recycling [40,52,53]. assisted endocytosis [1].Interestingly, The internalized experimental complex evidence is sorted suggests to thatendosomal there are compartments significant differences in where the ligand isthe released recycling and of LDLR LDLR complexed is recycled with to ApoE3the plasma or ApoE4 membrane [54–57]. However,[42–44]. Mul a mechanistic‐ tiple sources of evidenceexplanation demonstrate for the differences that LDLR in ApoE3- recycling and ApoE4-containingrequires organized complexes cellular is ac lacking.‐ To tions including luminalexplain endosomal these findings, acidification we propose thatand the calcium observed (Ca differences2+) signaling are due [45–51]. to the In differences particular, endosomalin the acidification pH-dependence plays of the a association key role betweenin the control ApoE3 andof LDLR ApoE4 recycling isoforms and LDL- [40,52,53]. A repeats of the LDLR. As a result, ApoE4-containing but not ApoE3-containing LDLR complexes are trapped in late endosomes and targeted for degradation, leading to a Interestingly,reduction experimental in the evidence steady state suggests surface that levels there of LDLRs are significant and impaired differences cellular signaling in in the recycling of LDLRApoE4-expressing complexed with cells. ApoE3 or ApoE4 [54–57]. However, a mechanistic explanation for the differences in ApoE3‐ and ApoE4‐containing complexes is lacking. To explain these findings, we propose that the observed differences are due to the differences in the pH‐dependence of the association between ApoE3 and ApoE4 isoforms and LDL‐ A repeats of the LDLR. As a result, ApoE4‐containing but not ApoE3‐containing LDLR complexes are trapped in late endosomes and targeted for degradation,