RESUMEN
Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid ß in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110-117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.
Asunto(s)
Anticuerpos Monoclonales , Apolipoproteínas E , Isoformas de Proteínas , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Humanos , Isoformas de Proteínas/inmunología , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/química , Apolipoproteínas E/inmunología , Animales , Epítopos/inmunología , Epítopos/química , Ensayo de Inmunoadsorción Enzimática/métodos , Ratones , Apolipoproteína E4/genética , Apolipoproteína E4/inmunología , Apolipoproteína E4/metabolismo , Ratones Endogámicos BALB C , Apolipoproteína E3/inmunología , Apolipoproteína E3/genética , Apolipoproteína E3/química , Apolipoproteína E3/metabolismoRESUMEN
The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.
Asunto(s)
Apolipoproteína E4 , Apolipoproteínas E , Apolipoproteína E4/genética , Apolipoproteína E3/química , Apolipoproteína E2 , Conformación Proteica , Isoformas de Proteínas/metabolismoRESUMEN
High levels of 4-hydroxynonenal (HNE), arising from lipid peroxidation, and HNE-modified proteins have been identified in postmortem brains of ageing and Alzheimer's disease (AD) patients. The goal of this study is to understand the effect of HNE modification on the structure and function of recombinant apolipoprotein E3 (apoE3) and apolipoprotein E4 (apoE4), which play a critical role in brain cholesterol homeostasis. The two isoforms differ in a single amino acid at position 112: Cys in apoE3 and Arg in apoE4. Immunoblot with HNE-specific antibody indicates HNE modification of apoE3 and apoE4 with a major band at ~ 36 kDa, while LC-MS/MS revealed Michael addition at His140 (60-70% abundance) and His299 (3-5% abundance) in apoE3 and apoE4, and Cys112 adduct in apoE3 (75% abundance). Circular dichroism spectroscopy revealed no major differences in the overall secondary structure or helical content between unmodified and HNE-modified apoE. HNE modification did not affect their ability to promote cholesterol efflux from J774.1 macrophages. However, it led to a 3-fold decrease in their ability to bind lipids and 25-50% decrease in the ability of cerebral cortex endothelial cells to uptake lipoproteins bearing HNE-modified HNE-apoE3 or HNE-apoE4 as noted by fluorescence microscopy and flow cytometry. Taken together, the data indicate that HNE modification impairs lipid binding and cellular uptake of both isoforms, and that apoE3, bearing a Cys, offers a protective role by sequestering lipid peroxidation products that would otherwise cause indiscriminate damage to biomolecules. ApoE4, lacking Cys, is unable to protect against oxidative damage that is commensurate with ageing.
Asunto(s)
Apolipoproteína E4 , Células Endoteliales , Humanos , Apolipoproteína E3/química , Apolipoproteína E4/química , Células Endoteliales/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Apolipoproteínas E/química , Colesterol , Isoformas de Proteínas/genéticaRESUMEN
Apolipoprotein E (ApoE) is one of the major lipid transporters in humans. It is also implicated in pathological conditions like Alzheimer's and cardiovascular diseases. The N-terminal domain of ApoE binds low-density lipoprotein receptors (LDLR) while the C-terminal domain binds to the lipid. I report the backbone and aliphatic side-chain NMR chemical shifts of the N-terminal domain of two isoforms of ApoE, namely ApoE3 NTD (BMRB No. 51,122) and ApoE4 NTD (BMRB No. 51,123) at pH 3.5 (20 °C).
Asunto(s)
Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteína E3/química , Apolipoproteína E3/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Humanos , Lipoproteínas LDL , Resonancia Magnética Nuclear Biomolecular , Isoformas de ProteínasRESUMEN
Apolipoprotein E (apoE) (299 residues) is a highly helical protein that plays a critical role in cholesterol homeostasis. It comprises a four-helix bundle N-terminal (NT) and a C-terminal (CT) domain that can exist in lipid-free and lipid-associated states. In humans, there are two major apoE isoforms, apoE3 and apoE4, which differ in a single residue in the NT domain, with apoE4 strongly increasing risk of Alzheimer's disease (AD) and cardiovascular diseases (CVD). It has been proposed that the CT domain initiates rapid lipid binding, followed by a slower NT domain helix bundle opening and lipid binding to yield discoidal reconstituted high density lipoprotein (rHDL). However, the contribution of the NT domain on the CT domain organization in HDL remains poorly understood. To understand this, we employed Cys-specific cross-linking and spatially-sensitive fluorophores in the NT and CT domains of apoE3 and apoE4, and in isolated CT domain. We noted that the helices in isolated CT domain are oriented parallel to those in the neighboring molecule in rHDL, whereas full length apoE3 and apoE4 adopt either an anti-parallel or hairpin-like organization. It appears that the bulky NT domain determines the spatial organization of its CT domain in rHDL, a finding that has significance for apoE4, which is more susceptible to proteolytic cleavage in AD brains, showing increased accumulation of neurotoxic NT and CT fragments. We envisage that the structural organization of HDL apoE would have profound functional consequences in its ability to regulate cholesterol homeostasis in AD and CVD.
Asunto(s)
Apolipoproteínas E/metabolismo , Lipoproteínas HDL/metabolismo , Apolipoproteína E3/química , Apolipoproteína E3/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Apolipoproteínas E/química , Apolipoproteínas E/genética , Lipoproteínas HDL/química , Dominios Proteicos , Estructura Secundaria de Proteína , Pirenos/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de FluorescenciaRESUMEN
Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.
Asunto(s)
Apolipoproteína E3/química , Dimiristoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , HumanosRESUMEN
PURPOSE: KRAS is the most frequently mutated gene in human cancers, and ~ 90% of pancreatic cancers exhibit KRAS mutations. Despite the well-known role of KRAS in malignancies, directly inhibiting KRAS is challenging. METHODS: In this study, we successfully synthesized apolipoprotein E3-based liposomes for the co-delivery of gemcitabine (GEM) and a small interfering RNA targeting KRAS (KRAS-siRNA) to improve the efficacy of pancreatic cancer treatment. RESULTS: Apolipoprotein E3 self-assembly on the liposome surface led to a substantial increase in its internalization in PANC1 human pancreatic cancer cells. KRAS-siRNA led to downregulated KRAS protein expression and KRAS-dependent carcinogenic pathways, resulting in the inhibition of cell proliferation, cell cycle arrest, increased apoptosis, and suppression of tumor progression. The combination of KRAS-siRNA and GEM induced a synergistic improvement in cell apoptosis and significantly lower cell viability compared with single-agent therapy. The low IC50 value of A3-SGLP might be attributed to potentiation of the anticancer effect of GEM by siRNA-mediated silencing of KRAS mutations, thereby inducing synergistic effects on cancer cells. CONCLUSION: A3-SGLP led to a marked decrease in the overall tumor burden and did not show any signs of toxicity. Therefore, the combination of KRAS-siRNA and GEM holds great potential for the treatment of pancreatic cancer.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apolipoproteína E3/metabolismo , Desoxicitidina/análogos & derivados , Técnicas de Transferencia de Gen , Lípidos/química , Nanopartículas , Neoplasias Pancreáticas/terapia , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Interferente Pequeño/metabolismo , Tratamiento con ARN de Interferencia , Animales , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/metabolismo , Apolipoproteína E3/química , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Terapia Combinada , Desoxicitidina/química , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Composición de Medicamentos , Humanos , Liposomas , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/genética , Receptores de LDL/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
ApoE4(C112R) is the strongest risk factor for Alzheimer's disease, while apoE3(C112) is considered normal. The C112R substitution is believed to alter the interactions between the N-terminal (NTD) and the C-terminal domain (CTD) leading to major functional differences. Here we investigate how the molecular property of the residue at position 112 affects domain interaction using an array of C112X substitutions with arginine, alanine, threonine, valine, leucine and isoleucine as 'X'. We attempt to determine the free energy of domain interaction (∆GINT) from stabilities of the NTD (∆GNTD) and CTD (∆GCTD) in the full-length apoE, and the stabilities of fragments of the NTD (∆GNTF) and CTD (∆GCTF), using the relationship, ∆GINT = ∆GNTD + ∆GCTD - ∆GNTF - ∆GCTF. We find that although ∆GNTD is strongly dependent on the C112X substitutions, ∆GNTD - ∆GNTF is small. Furthermore, ∆GCTD remains nearly the same as ∆GCTF. Therefore, ∆GINT is estimated to be small and similar for the apoE isoforms. However, stability of domain interaction monitored by urea dependent changes in interdomain Forster Resonance Energy Transfer (FRET) is found to be strongly dependent on C112X substitutions. ApoE4 exhibits the highest mid-point of denaturation of interdomain FRET. To resolve the apparently contradictory observations, we hypothesize that higher interdomain FRET in apoE4 in urea may involve 'intermediate' states. Enhanced fluorescence of bis-ANS and susceptibility to proteolytic cleavage support that apoE4, specifically, the NTD of apoE4 harbor 'intermediates' in both native and mildly denaturing conditions. The intermediates could hold key to the pathological functions of apoE4.
Asunto(s)
Apolipoproteína E4/química , Dominios y Motivos de Interacción de Proteínas , Apolipoproteína E3/química , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Isoformas de Proteínas , Estabilidad Proteica , Desplegamiento Proteico , Relación Estructura-ActividadRESUMEN
Although the interaction of apoE isoforms with amyloid-ß (Aß) peptides plays a critical role in the progression of Alzheimer's disease, how they interact with each other remains poorly understood. Here, we investigate the molecular mechanism of apoE-Aß interactions by comparing the effects of the different domains of apoE on Aß. The kinetics of aggregation of Aß1-42 are delayed dramatically in the presence of substoichiometric, nanomolar concentrations of N-terminal fragment (NTF), C-terminal fragment (CTF) and full-length apoE both in lipid-free and in lipidated forms. However, interactions between apoE and Aß as measured by intermolecular Förster resonance energy transfer (FRET) analysis were found to be minimal at t = 0 but to increase in a time-dependent manner. Thus, apoE must interact with one or more 'intermediates' rather than the monomers of Aß. Kinetics of FRET between full-length apoE4 labelled with EDANS at position 62 or 139 or 210 or 247 or 276, and tetramethylrhodamine-labelled Aß (TMR-Aß), further support an involvement of all the three domains of apoE in the interactions. However, the above-mentioned residues do not appear to form a single pocket in the 3-dimensional structure of apoE. A competitive binding assay examining the effects of unlabelled fragments or full-length apoE on the FRET between EDANS-apoE and TMR-Aß show that binding affinity of the full-length apoE to Aß is much higher than that of the fragments. Furthermore, apoE4 is found to interact more strongly than apoE3. We hypothesize that high affinity of the apoE-Aß interaction is attained due to multivalent binding mediated by multiple interactions between oligomeric Aß and full-length apoE.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Péptidos beta-Amiloides/química , Animales , Apolipoproteína E3/química , Apolipoproteína E3/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Apolipoproteínas E/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión ProteicaRESUMEN
Apolipoprotein E (apoE) is a 299 residue, exchangeable apolipoprotein that has essential roles in cholesterol homeostasis and reverse cholesterol transport. It is a two-domain protein with the C-terminal (CT) domain mediating protein self-association via helix-helix interactions. In humans, the APOE gene is polymorphic with three common alleles, ε2, ε3, and ε4, occurring in frequencies of ~ 5%, 77%, and 18%, respectively. Heterozygotes expressing apoE3 and apoE4 isoforms, which differ in residue at position 112 in the N-terminal domain (C112 in apoE3 and R112 in apoE4), represent the highest population of ε4 carriers, an allele highly associated with Alzheimer's disease. The objective of this study was to determine if apoE3 and apoE4 have the ability to hybridize to form a heteromer in lipid-free state. Refolding an equimolar mixture of His-apoE3 and FLAG-apoE4 (or vice versa) followed by pull-down and immunoblotting indicated formation of apoE3/apoE4 heteromers. Förster resonance energy transfer between donor fluorophore on one isoform and acceptor on the other, both located in the respective CT domains, revealed a distance of separation of ~ 46 Å between the donor/acceptor pair. Similarly, a quencher placed on one was able to mediate significant quenching of fluorescence emission on the other, indicative of spatial proximity within collisional distance between the two. ApoE3/apoE4 heteromer association was also noted in lipid-associated state in reconstituted lipoprotein particles. The possibility of heteromerization of apoE3/apoE4 bears implications in the potential mitigating role of apoE3 on the folding and physiological behavior of apoE4 and its role in maintaining cholesterol homeostasis.
Asunto(s)
Apolipoproteína E3/química , Apolipoproteína E3/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Dicroismo Circular , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Humanos , Fosfatidilcolinas/química , Conformación Proteica , Desnaturalización Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Among various isoforms of Apolipoprotein E (ApoE), the E4 isoform (ApoE4) is considered to be the strongest risk factor for Alzheimer's disease, whereas the E3 isoform (ApoE3) is neutral to the disease. Interestingly, the sequence of ApoE4 differs from its wild-type ApoE3 by a single amino acid C112R in the 299-amino-acid-long sequence. Hence, the puzzle remains: how a single-amino-acid difference between the ApoE3 and ApoE4 sequences can give rise to structural dissimilarities between the two isoforms, which can potentially lead to functional differences with significant pathological consequences. The major obstacle in addressing this question has been the lack of a 3D atomistic structure of ApoE4 to date. In this work, we resolve the issue by computationally modeling a plausible atomistic 3D structure of ApoE4. Our microsecond-long atomistic simulations elucidate key structural differences between monomeric ApoE3 and ApoE4, which renders ApoE4 thermodynamically less stable, less structured, and topologically less rigid compared to ApoE3. Consistent with an experimental report of the molten globule state of ApoE4, simulations identify multiple partially folded intermediates for ApoE4, which are implicated in the stronger aggregation propensity of ApoE4.
Asunto(s)
Apolipoproteína E3/química , Apolipoproteína E4/química , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Enlace de Hidrógeno , Cadenas de Markov , Simulación de Dinámica Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estabilidad Proteica , Estructura Secundaria de Proteína , TemperaturaRESUMEN
We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 µM for the three different regions, giving an overall binding affinity of 7.4 µM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.
Asunto(s)
Apolipoproteína E3/metabolismo , Péptidos/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Apolipoproteína E3/química , Sitios de Unión , Deuterio/química , Medición de Intercambio de Deuterio , Ligandos , Método de Montecarlo , Péptidos/química , Unión Proteica , Desplegamiento Proteico , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
PURPOSE: APOE ε7 gene is a rare mutant form of APOE ε3. The mutation occurs in the lipid-binding domain of APOE. Based on the protein's structure, APOE ε7 is expected to function in lipid and ß-amyloid metabolism, similar to APOE ε4. However, unlike that for APOE ε4, the mechanisms responsible for Alzheimer's disease (AD) cases associated with APOE ε7 expression have not been elucidated. The present study aims to investigate the association between APOE ε7 expression and cognitive impairment. METHODS: APOE was sequenced in DNA samples collected from 344 memory-complaint patients who visited the memory clinic, and from 345 non-memory-complaint individuals from the health promotion center. The protein structures of ApoE3, ApoE4, and ApoE7 were predicted. RESULTS: Three ε3/ε7 heterozygote individuals who were all classified under the memory-complaint group were identified. Of these, two subjects were clinically diagnosed with AD with small vessel disease, and the remaining individual was diagnosed with subjective cognitive impairment. This study predicted the protein structures of ApoE3, ApoE4, and ApoE7 and determined the three-dimensional structure of the carboxy terminus of ApoE7, which participates in an electrostatic domain interaction similar to that of APOE ε4. APOE K244 or K245 mutations for APOE ε7 were not found in the Korean reference genome database, which contains information (http://152.99.75.168/KRGDB/browser/mainBrowser.jsp) from 622 healthy individuals. CONCLUSION: As verified by the results of structural prediction, APOE ε7 could serve as another risk factor for cognitive impairment and is particularly associated with vascular disease. However, additional studies are required to validate the pathogenic nature of APOE ε7.
Asunto(s)
Apolipoproteína E3/genética , Apolipoproteínas E/genética , Disfunción Cognitiva/genética , Anciano , Alelos , Enfermedad de Alzheimer/genética , Apolipoproteína E3/química , Apolipoproteína E4/química , Apolipoproteína E4/genética , Femenino , Humanos , Masculino , Memoria , Persona de Mediana Edad , Solución de Problemas , República de Corea , Factores de RiesgoRESUMEN
Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues) is composed of an N-terminal (NT) domain bearing a 4-helix bundle and a C-terminal (CT) domain bearing a series of amphipathic α-helices. ApoAI (243 residues) also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function.
Asunto(s)
Apolipoproteína A-I/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína A-I/química , Apolipoproteína E3/química , Línea Celular Tumoral , Cromatografía de Afinidad , Dicroismo Circular , Dimiristoilfosfatidilcolina/química , Escherichia coli , Glioblastoma/metabolismo , Humanos , Immunoblotting , Macrófagos/metabolismo , Espectrometría de Masas , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Desplegamiento Proteico , Receptores de LDL/metabolismo , Espectrometría de Fluorescencia , Relación Estructura-Actividad , TransfecciónRESUMEN
Relative to the apolipoprotein E (apoE) E3 allele of the APOE gene, apoE4 strongly increases the risk for the development of late-onset Alzheimer's disease. However, apoE4 differs from apoE3 by only a single amino acid at position 112, which is arginine in apoE4 and cysteine in apoE3. It remains unclear why apoE3 and apoE4 are functionally different. Described here is a proposal for understanding the functional differences between these two isoforms with respect to lipid binding. A mechanism is proposed that is based on the full-length monomeric structure of the protein, on hydrogen-deuterium exchange mass spectrometry data, and on the role of intrinsically disordered regions to control protein motions. It is proposed that lipid binds between the N-terminal and C-terminal domains and that separation of the two domains, along with the presence of intrinsically disordered regions, controls this process. The mechanism explains why apoE3 differs from apoE4 with respect to different lipid-binding specificities, why lipid increases the binding of apoE to its receptor, and why specific residues are conserved.
Asunto(s)
Apolipoproteína E3/química , Apolipoproteína E3/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Metabolismo de los Lípidos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Sitios de Unión/genética , Fenómenos Biofísicos , Secuencia Conservada , Medición de Intercambio de Deuterio , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
ApoE3 is the major chylomicron apolipoprotein, binding in a specific liver peripheral cell receptor, allowing transport and normal catabolism of triglyceride-rich lipoprotein constituents. Point mutations in ApoE3 have been associated with Alzheimer's disease, type III hyperlipoproteinemia, atherosclerosis, telomere shortening and impaired cognitive function. Here, we evaluate the impact of missense SNPs in APOE retrieved from dbSNP through 16 computational prediction tools, and further evaluate the structural impact of convergent deleterious changes using 100 ns molecular dynamics simulations. We have found structural changes in four analyzed variants (Pro102Arg, Arg132Ser, Arg176Cys and Trp294Cys), two of them (Pro102Arg and Arg176Cys) being previously associated with human diseases. In all cases, except for Trp294Cys, there was a loss in the number of hydrogen bonds between CT and NT domains that could result in their detachment. In conclusion, data presented here could increase the knowledge of ApoE3 activity and be a starting point for the study of the impact of variations on APOE gene.
Asunto(s)
Apolipoproteína E3/genética , Mutación Missense/genética , Polimorfismo de Nucleótido Simple/genética , Relación Estructura-Actividad , Apolipoproteína E3/química , Apolipoproteínas E/química , Apolipoproteínas E/genética , Simulación por Computador , Humanos , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
BACKGROUND: Apolipoprotein E (apoE) is closely involved in the pathogenesis of apoE-related diseases, such as Alzheimer's disease and cardiovascular disease. The redox modulation of cysteine-thiols in a protein is involved in various pathophysiological regulations; however, that of apoE has not been studied in detail. Herein, we devised an analytical method to determine the redox status of serum apoE and assessed its relation to serum cholesterol levels and apoE phenotype. METHODS: The present method was based on a band shift assay, using a photocleavable maleimide-conjugated polyethylene glycol. RESULTS: The basic characteristics of the present method were found to be satisfactory to determine the redox status of serum apoE quantitatively. Serum apoE was separated into its reduced-form (r-), non-reduced-form (nr-), apoE-AII complex, and homodimer using this method. R-apoE could be detected as a 40-kDa band, whereas nr-apoE remained as monomeric apoE. R-apoE displayed a preference for VLDL; however, the levels showed the correlation with HDL-cholesterol levels (p<0.005). Redox status of serum apoE was significantly different among apoE phenotypes. The quantitative ratios of nr-apoE to total apoE in serum from subjects with apoE4/E3 were higher than in serum from subjects with apoE3/E3 (p<0.0001) and apoE3/E2 (p<0.001). CONCLUSION: The redox status of serum apoE might be related to the synthesis of HDL. The information concerning the redox status of serum apoE provided by the present method may be a potent indicator to evaluate various apoE-related diseases.
Asunto(s)
Apolipoproteínas E/sangre , HDL-Colesterol/sangre , Apolipoproteína A-II/sangre , Apolipoproteína A-II/química , Apolipoproteína A-II/aislamiento & purificación , Apolipoproteína E2/sangre , Apolipoproteína E2/química , Apolipoproteína E2/aislamiento & purificación , Apolipoproteína E3/sangre , Apolipoproteína E3/química , Apolipoproteína E3/aislamiento & purificación , Apolipoproteína E4/sangre , Apolipoproteína E4/química , Apolipoproteína E4/aislamiento & purificación , Apolipoproteínas E/química , Apolipoproteínas E/aislamiento & purificación , HDL-Colesterol/química , Cisteína/química , Diamida/química , Dimerización , Ditiotreitol/química , Ensayo de Cambio de Movilidad Electroforética , Células HEK293 , Humanos , Indicadores y Reactivos/química , Peso Molecular , Oxidación-Reducción , Procesos Fotoquímicos , Polietilenglicoles/química , Solubilidad , Reactivos de Sulfhidrilo/química , Rayos UltravioletaRESUMEN
Apolipoprotein E (apoE) plays a critical role in cholesterol transport in both peripheral circulation and brain. Human apoE is a polymorphic 299-residue protein in which the less common E4 isoform differs from the major E3 isoform only by a C112R substitution. ApoE4 interacts with lipoprotein particles and with the amyloid-ß peptide, and it is associated with increased incidence of cardiovascular and Alzheimer's disease. To understand the structural basis for the differences between apoE3 and E4 functionality, we used hydrogen-deuterium exchange coupled with a fragment separation method and mass spectrometric analysis to compare their secondary structures at near amino acid resolution. We determined the positions, dynamics, and stabilities of the helical segments in these two proteins, in their normal tetrameric state and in mutation-induced monomeric mutants. Consistent with prior X-ray crystallography and NMR results, the N-terminal domain contains four α-helices, 20 to 30 amino acids long. The C-terminal domain is relatively unstructured in the monomeric state but forms an α-helix â¼70 residues long in the self-associated tetrameric state. Helix stabilities are relatively low, 4 kcal/mol to 5 kcal/mol, consistent with flexibility and facile reversible unfolding. Secondary structure in the tetrameric apoE3 and E4 isoforms is similar except that some helical segments in apoE4 spanning residues 12 to 20 and 204 to 210 are unfolded. These conformational differences result from the C112R substitution in the N-terminal helix bundle and likely relate to a reduced ability of apoE4 to form tetramers, thereby increasing the concentration of functional apoE4 monomers, which gives rise to its higher lipid binding compared with apoE3.
Asunto(s)
Apolipoproteína E3/química , Apolipoproteína E4/química , Espectrometría de Masas/métodos , Sustitución de Aminoácidos , Apolipoproteína E4/genética , Dicroismo Circular , Predisposición Genética a la Enfermedad , Hidrógeno/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Lipoproteínas/metabolismo , Mutación Missense , Mutación Puntual , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/químicaRESUMEN
Apolipoprotein (apo) E was initially described as a lipid transport protein and major ligand for low density lipoprotein (LDL) receptors with a role in cholesterol metabolism and cardiovascular disease. It has since emerged as a major risk factor (causative gene) for Alzheimer's disease and other neurodegenerative disorders. Detailed understanding of the structural features of the three isoforms (apoE2, apoE3, and apoE4), which differ by only a single amino acid interchange, has elucidated their unique functions. ApoE2 and apoE4 increase the risk for heart disease: apoE2 increases atherogenic lipoprotein levels (it binds poorly to LDL receptors), and apoE4 increases LDL levels (it binds preferentially to triglyceride-rich, very low density lipoproteins, leading to downregulation of LDL receptors). ApoE4 also increases the risk for neurodegenerative diseases, decreases their age of onset, or alters their progression. ApoE4 likely causes neurodegeneration secondary to its abnormal structure, caused by an interaction between its carboxyl- and amino-terminal domains, called domain interaction. When neurons are stressed or injured, they synthesize apoE to redistribute cholesterol for neuronal repair or remodeling. However, because of its altered structure, neuronal apoE4 undergoes neuron-specific proteolysis, generating neurotoxic fragments (12-29 kDa) that escape the secretory pathway and cause mitochondrial dysfunction and cytoskeletal alterations, including tau phosphorylation. ApoE4-associated pathology can be prevented by small-molecule structure correctors that block domain interaction by converting apoE4 to a molecule that resembles apoE3 both structurally and functionally. Structure correctors are a potential therapeutic approach to reduce apoE4 pathology in both cardiovascular and neurological disorders.
Asunto(s)
Apolipoproteína E2/genética , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Enfermedades Cardiovasculares/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas tau/genética , Apolipoproteína E2/metabolismo , Apolipoproteína E3/química , Apolipoproteína E3/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/metabolismo , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/patología , Colesterol/metabolismo , Regulación de la Expresión Génica , Humanos , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Modelos Moleculares , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción de Señal , Proteínas tau/metabolismoRESUMEN
PURPOSE: The present investigation aimed at brain targeting of sumatriptan succinate (SS) for its optimal therapeutic effect in migraine through nanoparticulate drug delivery system using poly (butyl cyanoacrylate) (PBCA) and bovine serum albumin linked with apolipoprotein E3 (BSA-ApoE). METHOD: The study involved formulation optimization of PBCA nanoparticles (NPs) using central composite design for achieving minimum particle size, maximum entrapment efficiency along with sustained drug release. SS incorporated in BSA-ApoE NPs (S-AA-NP) were prepared by desolvation technique and compared with SS loaded polysorbate 80 coated optimized PBCA NPs (FPopt) in terms of their brain uptake potential, upon oral administration in male Wistar rats. The NPs were characterized by FTIR, thermal, powder XRD and TEM analysis. RESULTS: The in vivo studies of FPopt and S-AA-NP on male Wistar rats demonstrated a fairly high brain/plasma drug ratio of 9.45 and 12.67 respectively 2 h post oral drug administration. The behavioural studies on male Swiss albino mice affirmed the enhanced anti-migraine potential of S-AA-NP than FPopt (P < 0.001). CONCLUSION: The results of this work, therefore, indicate that BSA-ApoE NPs are significantly better than polysorbate 80 coated PBCA NPs for brain targeting of SS (P < 0.05) and also offer an improved therapeutic strategy for migraine management.