RESUMO
Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.
Assuntos
Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste , Receptores de LDL , Receptores de LDL/metabolismo , Receptores de LDL/química , Vírus da Encefalite Equina do Leste/metabolismo , Vírus da Encefalite Equina do Leste/ultraestrutura , Humanos , Animais , Vírus da Floresta de Semliki/metabolismo , Ligação Proteica , Receptores Virais/metabolismo , Receptores Virais/química , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/ultraestrutura , Modelos MolecularesRESUMO
Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia-associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9-targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9-LDL receptor interaction, thereby attenuating the PCSK9-mediated impairment of LDL cholesterol clearance. The N-terminal sequence of human H ferritin was engineered to incorporate a 13-amino acid linear peptide (Pep2-8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9-binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9-LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin-based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin-based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.
Assuntos
LDL-Colesterol , Nanopartículas , Pró-Proteína Convertase 9 , Receptores de LDL , Humanos , Pró-Proteína Convertase 9/metabolismo , Pró-Proteína Convertase 9/química , Pró-Proteína Convertase 9/genética , Receptores de LDL/metabolismo , Receptores de LDL/química , Nanopartículas/química , LDL-Colesterol/metabolismo , Inibidores de PCSK9/farmacologia , Inibidores de PCSK9/química , Ferritinas/química , Ferritinas/metabolismo , Ligação ProteicaRESUMO
The protein PCSK9 (proprotein convertase subtilisin/Kexin type 9) negatively regulates the recycling of LDLR (low-density lipoprotein receptor), leading to an elevated plasma level of LDL. Inhibition of PCSK9-LDLR interaction has emerged as a promising therapeutic strategy to manage hypercholesterolemia. However, the large interaction surface area between PCSK9 and LDLR makes it challenging to identify a small molecule competitive inhibitor. An alternative strategy would be to identify distal cryptic sites as targets for allosteric inhibitors that can remotely modulate PCSK9-LDLR interaction. Using several microseconds long molecular dynamics (MD) simulations, we demonstrate that on binding with LDLR, there is a significant conformational change (population shift) in a distal loop (residues 211-222) region of PCSK9. Consistent with the bidirectional nature of allostery, we establish a clear correlation between the loop conformation and the binding affinity with LDLR. Using a thermodynamic argument, we establish that the loop conformations predominantly present in the apo state of PCSK9 would have lower LDLR binding affinity, and they would be potential targets for designing allosteric inhibitors. We elucidate the molecular origin of the allosteric coupling between this loop and the LDLR binding interface in terms of the population shift in a set of salt bridges and hydrogen bonds. Overall, our work provides a general strategy toward identifying allosteric hotspots: compare the conformational ensemble of the receptor between the apo and bound states of the protein and identify distal conformational changes, if any. The inhibitors should be designed to bind and stabilize the apo-specific conformations.
Assuntos
Simulação de Dinâmica Molecular , Pró-Proteína Convertase 9 , Ligação Proteica , Receptores de LDL , Receptores de LDL/metabolismo , Receptores de LDL/química , Pró-Proteína Convertase 9/metabolismo , Pró-Proteína Convertase 9/química , Regulação Alostérica , Humanos , Conformação Proteica , Termodinâmica , Inibidores de PCSK9RESUMO
The complex structure and function of low density lipoprotein receptor (LDLR) makes classification of protein-coding missense variants challenging. Deep generative models, including Evolutionary model of Variant Effect (EVE), Evolutionary Scale Modeling (ESM), and AlphaFold 2 (AF2), have enabled significant progress in the prediction of protein structure and function. ESM and EVE directly estimate the likelihood of a variant sequence but are purely data-driven and challenging to interpret. AF2 predicts LDLR structures, but variant effects are explicitly modeled by estimating changes in stability. We tested the effectiveness of these models for predicting variant pathogenicity compared to established methods. AF2 produced two distinct conformations based on a novel hinge mechanism. Within ESM's hidden space, benign and pathogenic variants had different distributions. In EVE, these distributions were similar. EVE and ESM were comparable to Polyphen-2, SIFT, REVEL, and Primate AI for predicting binary classifications in ClinVar. However, they were more strongly correlated with experimental measures of LDL uptake. AF2 poorly performed in these tasks. Using the UK Biobank to compare association with clinical phenotypes, ESM and EVE were more strongly associated with serum LDL-C than Polyphen-2. ESM was able to identify variants with more extreme LDL-C levels than EVE and had a significantly stronger association with atherosclerotic cardiovascular disease. In conclusion, AF2 predicted LDLR structures do not accurately model variant pathogenicity. ESM and EVE are competitive with prior scoring methods for prediction based on binary classifications in ClinVar but are superior based on correlations with experimental assays and clinical phenotypes.
Assuntos
Modelos Moleculares , Receptores de LDL , Virulência , Receptores de LDL/química , Receptores de LDL/genética , Estrutura Terciária de Proteína , Variação Genética , Virulência/genética , Fenótipo , Humanos , Doenças Cardiovasculares/fisiopatologiaRESUMO
BACKGROUND: Familial hypercholesterolemia (FH) is a globally underdiagnosed inherited metabolic disorder. Owing to limited published data from Arab world, this study was conducted with the aim of identifying the genetic and molecular basis of FH in highly consanguineous Saudi population. METHODS: We performed clinical screening, biochemical profiling, whole exome sequencing and variant segregation analysis of two Saudi FH families. Additionally, 500 normolipic individuals were screened to ensure the absence of FH variant in general Saudi population. Functional characterization of FH variants on secondary structure characteristics of RNA and protein molecules was performed using different bioinformatics modelling approaches. RESULTS: WES analysis identified two independent rare LDLR gene stop gain variants (p.C231* and p.R744*) consistent to the clinical presentation of FH patients from two different families. RNAfold analysis has shown that both variants were predicted to disturb the free energy dynamics of LDLR mRNA molecule and destabilize its folding pattern and function. PSIPRED based structural modelling analysis has suggested that both variants bring drastic changes disturbing the secondary structural elements of LDLR molecule. The p.C231* and p.R744* variants are responsible for partial or no protein product, thus they are class 1 variants causing loss of function (LoF) LDLR variants. CONCLUSIONS: This study highlights the effectiveness of the WES, sanger sequencing, and computational analysis in expanding FH variant spectrum in culturally distinct populations like Saudi Arabia. Genetic testing of FH patients is very essential in better clinical diagnosis, screening, treatment, and management and prevention of cardiovascular disease burden in the society.
Assuntos
Doenças Cardiovasculares , Hiperlipoproteinemia Tipo II , Humanos , Testes Genéticos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/epidemiologia , Mutação , Fenótipo , Receptores de LDL/genética , Receptores de LDL/química , Receptores de LDL/metabolismo , Arábia SauditaRESUMO
BACKGROUND: Familial hypercholesterolemia (FH) is a common illness mainly caused by variants occurring in the low-density lipoprotein receptor (LDLR) gene. FH is a leading cause of coronary artery disease. OBJECTIVE: This study aims to determine genetic defect(s) in homozygous and heterozygous FH index patients and their first-degree blood relatives and understand the genotype-phenotype correlation. METHODS: This study employed the genetic screening of FH-related genes by next-generation sequencing and cascade screening by capillary sequencing. RESULTS: We identified the presence of a novel frameshift variant [c.335_336insCGAG, p.(F114Rfs*17)] and three known missense variants [c.622G>A, p.(E208K)], [c.1474G>A, p.(D492N)], [c.1429G>A, p.(D477N)] in the LDLR gene of four unrelated Saudi families with FH. In proband 1, a nonsense variant c.1421C>G, p.(S474*) was also detected at exon 9 of the lipoprotein lipase gene. The segregation arrangement of the identified variants corresponded with the clinical characteristics. In this study, all the detected variants were confined in the ligand-binding domain and epidermal growth factor (EGF)-precursor homology domain of the LDLR protein, which portrayed severe clinical phenotypes of FH. Moreover, these LDLR variants were in a highly conserved residue of the proteins. CONCLUSION: In addition to the finding of the novel variant in the LDLR gene that extends the spectrum of variants causing FH, the results of this study also support the need for diagnostic screening and cascade genetic testing of this high-risk condition and to understand the genotype-phenotype correlation, which could lead to better prevention of coronary artery disease.
Assuntos
Doença da Artéria Coronariana , Hiperlipoproteinemia Tipo II , Humanos , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/genética , Arábia Saudita/epidemiologia , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/epidemiologia , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Receptores de LDL/química , Homozigoto , Fenótipo , MutaçãoRESUMO
In this work, we designed three new ligands by conjugating cholesterol metabolites 3-hydroxy-5-cholestenoic acid (3-HC) and 3-oxo-4-cholestenoic acid (3-OC) and the natural tri-terpenoid betulinic acid with the tumor-targeting peptide YHWYGYTPQNVI. Molecular interactions with the unconjugated peptide and the conjugates were examined with three receptors that are commonly overexpressed in pancreatic adenocarcinoma cells using ligand docking and molecular dynamics. This study demonstrated the utility of the designed conjugates as a valuable scaffold for potentially targeting EGFR and LDLR receptors. Our results indicate that the conjugates showed strong binding affinities and formation of stable complexes with EGFR, while the unconjugated peptide, BT-peptide conjugate, an 3-HC-peptide conjugate showed the formation of fairly stable complexes with LDLR receptor. For EGFR, two receptor kinase domains were explored. Interactions with the N-terminal domain of CCKA-R were relatively weaker. For LDLR, binding occurred in the beta-propeller region. For the N-terminal fragment of CCKA-R, the conjugates induced significant conformational changes in the receptor. The molecular dynamic simulations for 100 ns demonstrate that BT-peptide conjugates and the unconjugated peptide had the highest binding and formed the most stable complexes with EGFR. RMSD and trajectory analyses indicate that these molecules transit to a dynamically stable configuration in most cases within 60 ns. NMA analysis indicated that amongst the conjugates that showed relatively higher interactions with the respective receptors, the highest potential for deformability was seen for the N-terminal-47 amino acid region of the CCKA-R receptor with and the lowest for the LDLR-receptor. Thus, the newly designed compounds may be evaluated in the future toward developing drug delivery materials for targeting tumor cells overexpressing LDLR or EGFR.
Assuntos
Colesterol/química , Modelos Moleculares , Triterpenos Pentacíclicos/química , Peptídeos/química , Receptor de Colecistocinina A/química , Receptores de LDL/química , Sequência de Aminoácidos , Receptores ErbB/química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Ácido BetulínicoRESUMO
Low plasma levels of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) are associated with decreased low-density lipoprotein (LDL) cholesterol and a reduced risk of cardiovascular disease. PCSK9 binds to the epidermal growth factor-like repeat A (EGFA) domain of LDL receptors (LDLR), very low-density lipoprotein receptors (VLDLR), apolipoprotein E receptor 2 (ApoER2), and lipoprotein receptor-related protein 1 (LRP1) and accelerates their degradation, thus acting as a key regulator of lipid metabolism. Antibody and RNAi-based PCSK9 inhibitor treatments lower cholesterol and prevent cardiovascular incidents in patients, but their high-cost hampers market penetration. We sought to develop a safe, long-term and one-time solution to treat hyperlipidemia. We created a cDNA encoding a chimeric protein in which the extracellular N- terminus of red blood cells (RBCs) specific glycophorin A was fused to the LDLR EGFA domain and introduced this gene into mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). Following transplantation into irradiated mice, the animals produced RBCs with the EGFA domain (EGFA-GPA RBCs) displayed on their surface. These animals showed significantly reduced plasma PCSK9 (66.5% decrease) and reduced LDL levels (40% decrease) for as long as 12 months post-transplantation. Furthermore, the EGFA- GPA mice remained lean for life and maintained normal body weight under a high-fat diet. Hematopoietic stem cell gene therapy can generate red blood cells expressing an EGFA-glycophorin A chimeric protein as a practical and long-term strategy for treating chronic hyperlipidemia and obesity.
Assuntos
LDL-Colesterol/sangue , Regulação para Baixo , Glicoforinas/genética , Hiperlipidemias/prevenção & controle , Pró-Proteína Convertase 9/sangue , Receptores de LDL/genética , Animais , Peso Corporal , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Eritrócitos/metabolismo , Feminino , Engenharia Genética , Glicoforinas/química , Células HEK293 , Humanos , Hiperlipidemias/induzido quimicamente , Hiperlipidemias/metabolismo , Camundongos , Gravidez , Receptores de LDL/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transplante de Células-Tronco , Transdução GenéticaRESUMO
Venezuelan equine encephalitis virus (VEEV) is an enveloped RNA virus that causes encephalitis and potentially mortality in infected humans and equines1. At present, no vaccines or drugs are available that prevent or cure diseases caused by VEEV. Low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3) was recently identified as a receptor for the entry of VEEV into host cells2. Here we present the cryo-electron microscopy structure of the LDLRAD3 extracellular domain 1 (LDLRAD3-D1) in complex with VEEV virus-like particles at a resolution of 3.0 Å. LDLRAD3-D1 has a cork-like structure and is inserted into clefts formed between adjacent VEEV E2-E1 heterodimers in the viral-surface trimer spikes through hydrophobic and polar contacts. Mutagenesis studies of LDLRAD3-D1 identified residues that are involved in the key interactions with VEEV. Of note, some of the LDLRAD3-D1 mutants showed a significantly increased binding affinity for VEEV, suggesting that LDLRAD3-D1 may serve as a potential scaffold for the development of inhibitors of VEEV entry. Our structures provide insights into alphavirus assembly and the binding of receptors to alphaviruses, which may guide the development of therapeutic countermeasures against alphaviruses.
Assuntos
Vírus da Encefalite Equina Venezuelana/química , Receptores de LDL/química , Receptores Virais/química , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Internalização do VírusRESUMO
LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus-receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.
Assuntos
Vírus da Encefalite Equina Venezuelana/química , Receptores de LDL/química , Receptores Virais/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Microscopia Crioeletrônica , Humanos , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Alinhamento de Sequência , Internalização do VírusRESUMO
Interaction of fibrin with the very low-density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation. To establish the structural basis for this interaction, we have previously localized the VLDLR-binding site to fibrin ßN-domains including fibrin ß chain sequence 15-64 and determined the NMR solution structure of the VLDLR(2-4) fragment containing fibrin-binding CR domains 2-4 of VLDLR. In this study, we identified amino acid residues in VLDLR and the ßN-domains that are involved in the interaction using NMR and site-directed mutagenesis. The results obtained revealed that Lys47 and Lys53 of the second and third positively charged clusters of the ßN-domain, respectively, interact with Trp20 and Asp25 of the CR2 domain and Trp63 and Glu68 of the CR3 domain, respectively. This finding indicates that Lys residues of the ßN-domain interact with the Lys-binding site of the CR domains in a manner proposed earlier for the interaction of other members of the LDL receptor family with their ligands. In addition, Gly15 of the ßN-domain and its first positively charged cluster contribute to the high-affinity interaction with VLDLR. Molecular modeling based on the results obtained and analysis of the previously published structures of such domains complexed with RAP and HRV2 allowed us to propose a model of interaction of fibrin ßN-domains with the fibrin-binding CR domains of the VLDL receptor.
Assuntos
Fibrina/química , Fibrina/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Mutagênese Sítio-Dirigida/métodos , Receptores de LDL/química , Receptores de LDL/metabolismo , Acetilação , Sítios de Ligação , Ensaio de Imunoadsorção Enzimática , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Receptores de LDL/genética , Ressonância de Plasmônio de SuperfícieRESUMO
The epidermal growth-factor-like domain A (EGF-A) of the low-density lipoprotein (LDL) receptor is a promising lead for therapeutic inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9). However, the clinical potential of EGF-A is limited by its suboptimal affinity for PCSK9. Here, we use phage display to identify EGF-A analogues with extended bioactive segments that have improved affinity for PCSK9. The most potent analogue, TEX-S2_03, demonstrated â¼130-fold improved affinity over the parent domain and had a reduced calcium dependency for efficient PCSK9 binding. Thermodynamic binding analysis suggests the improved affinity of TEX-S2_03 is enthalpically driven, indicating favorable interactions are formed between the extended segment of TEX-S2_03 and the PCSK9 surface. The improved affinity of TEX-S2_03 resulted in increased activity in competition binding assays and more efficient restoration of LDL receptor levels with clearance of extracellular LDL cholesterol in functional cell assays. These results confirm that TEX-S2_03 is a promising therapeutic lead for treating hypercholesterolemia. Many EGF-like domains are involved in disease-related protein-protein interactions; therefore, our strategy for engineering EGF-like domains has the potential to be broadly implemented in EGF-based drug design.
Assuntos
Fragmentos de Peptídeos/metabolismo , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismo , Sequência de Aminoácidos , Células Hep G2 , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Biblioteca de Peptídeos , Pró-Proteína Convertase 9/química , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Receptores de LDL/química , Receptores de LDL/genética , TermodinâmicaRESUMO
Apolipoprotein E (ApoE) is of great interest due to its role as a cholesterol/lipid transporter in the central nervous system (CNS) and as the most influential genetic risk factor for Alzheimer disease (AD). Work over the last four decades has given us important insights into the structure of ApoE and how this might impact the neuropathology and pathogenesis of AD. In this review, we highlight the history and progress in the structural and molecular understanding of ApoE and discuss how these studies on ApoE have illuminated the physiology of ApoE, receptor binding, and interaction with amyloid-ß (Aß). We also identify future areas of study needed to advance our understanding of how ApoE influences neurodegeneration.
Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Multimerização Proteica/fisiologia , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Receptores de LDL/química , Receptores de LDL/metabolismoRESUMO
There currently is no specific antiviral drug or a vaccine for SARS-CoV-2/COVID-19 infections; now exceeding 10,300,000 infections worldwide. In the absence of animal models to test drugs, we need to find molecular explanations for any unforeseen peculiarities in clinical data, especially the recent reports describing an unexpected asthma paradox. Asthma is considered a high medical risk factor for susceptibility to SARS-CoV-2/COVID-19 infection, yet asthma is not on the list of top 10 chronic health problems suffered by people who died from SARS-CoV-2/COVID-19. Resolving this paradox requires looking beyond the binary model of a viral receptor-binding domain (RBD) attaching to the ACE-2 receptor. A NCBI pBlast analysis revealed that the SARS-CoV-2 surface spike protein contains key two calcium-dependent fusion domains that are almost identical to those that were recently discovered SARS-CoV-1. These viral calcium-dependent binding domains can facilitate membrane fusion only after cleavage by the host surface protease TMPRSS2. Importantly, TMPRSS2 also requires calcium for its SRCR (scavenger receptor cysteine-rich) domain and itsLDLRA(LDL receptor class A) domain. Thus, the presence of EDTA excipients in nebulized ß2-agonist medicines can disrupt SARS-CoV-2/COVID-19 infection and can explain the asthma paradox. This model validates repurposing EDTA in nebulizer solutions from a passive excipient to an active drug for treating COVID-19 infections. Repurposed EDTA delivery to respiratory tissues at an initial target dose of 2.4 mg per aerosol treatment is readily achievable with standard nebulizer and mechanical ventilator equipment. EDTA warrants further investigation as a potential treatment for SARS-CoV-2/COVID-19 in consideration of the new calcium requirements for virus infection and the regular presence of EDTA excipients in common asthma medications such as Metaproterenol. Finally, the natural history of Coronavirus diseases and further analysis of the fusion loop homologies between the Betacorona SARS-CoV-2 virus and the less pathogenic Alphacorona HC0V-229E virus suggest how to engineer a hybrid virus suitable for an attenuated alpha-beta SARS-CoV-2/COVID-19 vaccine. Thus, replacing SARS-CoV-2 fusion loops (amino acids 816-855) with the less pathogenic HCoV-229E fusion loop (amino acids 923-982) may provide antigenicity of COVID-19, but limit the pathogenicity to the level of HCoV-229E.
Assuntos
Asma/complicações , Asma/epidemiologia , COVID-19/complicações , COVID-19/epidemiologia , Reposicionamento de Medicamentos , Ácido Edético/uso terapêutico , Antivirais/uso terapêutico , Broncodilatadores/uso terapêutico , Vacinas contra COVID-19 , Cálcio/química , Suscetibilidade a Doenças , Excipientes/uso terapêutico , Humanos , Metaproterenol/uso terapêutico , Modelos Teóricos , Nebulizadores e Vaporizadores , Prevalência , Receptores de LDL/química , Fatores de Risco , Serina Endopeptidases/metabolismoRESUMO
Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
Assuntos
Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Domínios FERM , Humanos , Modelos Moleculares , Receptores de LDL/química , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.
Assuntos
Retículo Endoplasmático/metabolismo , Receptores de LDL/química , Receptores de LDL/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Células HeLa , Humanos , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Receptores de LDL/metabolismoRESUMO
Gain-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to reduced uptake of LDL (low density lipoprotein) cholesterol and, therefore, increased plasma LDL levels. However, the mechanism by which these mutants reduce LDL reuptake is not fully understood. Here, we have used molecular dynamics simulations, MM/PBSA (Molecular Mechanics/Poisson-Boltzmann Surface Area) binding affinity calculations, and residue interaction networks, to investigate the protein-protein interaction (PPI) disruptive effects of two of PCSK9's gain-of-function mutations, Ser127Arg and Asp374Tyr on the PCSK9 and LDL receptor complex. In addition to these PPI disruptive mutants, a third, non-interface mutation (Arg496Trp) is included as a positive control. Our results indicate that Ser127Arg and Asp374Tyr confer significantly improved binding affinity, as well as different binding modes, when compared to the wild-type. These PPI disruptive mutations lie between the EGF(A) (epidermal growth factor precursor homology domain A) of the LDL receptor and the catalytic domain of PCSK9 (Asp374Tyr) and between the prodomain of PCSK9 and the ß-propeller of the LDL receptor (Ser127Arg). The interactions involved in these two interfaces result in an LDL receptor that is sterically inhibited from entering its closed conformation. This could potentially implicate the prodomain as a target for small molecule inhibitors.
Assuntos
Pró-Proteína Convertase 9/química , Receptores de LDL/química , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , Mutação , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Ligação Proteica , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is a primary hyperlipemia. It is an autosomal dominant genetic disorder of lipoproteins metabolism mainly caused by mutations in the low density lipoprotein receptor gene (LDLR). We aimed to investigate the functional impact on the low density lipoprotein receptor (LDLR) activity of six uncharacterised variants located in the coding region of the LDLR gene, namely c.428Gâ¯>â¯T, c.640Tâ¯>â¯C, c.1708Câ¯>â¯T, c.1736Aâ¯>â¯T, c.1981Câ¯>â¯G and c.2114Câ¯>â¯G (NM_000527.4) and to attempt to define their clinical status. METHODS: Functional studies were carried out using site-directed mutagenesis techniques and expression of LDLR protein in vitro. Results were correlated with clinical data and in silico analyses in order to assess the physiopathological role of these variants. RESULTS: This work provides functional information about 6 uncharacterised mutations in LDLR. CONCLUSIONS: The six variants studied here appeared to affect the LDLR function in vitro to different degrees, ranging from receptors with normal to slightly reduced activity to receptors exhibiting less than 10% of the wild-type activity. According to these studies and The American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines, two variants could be classified as "Likely Benign" (p.(Ala705Gly) and p.(Leu570Phe)), three variants as "Pathogenic" (p.(Asp579Val), p.(Cys143Phe) and p.(Trp214Arg)) and one variant as "Likely Pathogenic" (p.(Pro661Ala)).
Assuntos
Hiperlipoproteinemia Tipo II/genética , Metabolismo dos Lipídeos/genética , Mutação , Receptores de LDL/genética , Adulto , Idoso , Animais , Células CHO , Simulação por Computador , Cricetulus , Feminino , Predisposição Genética para Doença , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Fenótipo , Conformação Proteica , Receptores de LDL/química , Receptores de LDL/metabolismo , Medição de Risco , Fatores de Risco , Relação Estrutura-AtividadeRESUMO
Familial hypercholesterolemia (FH) is an autosomal dominant disease most often caused by mutations in the low-density lipoprotein receptor (LDLR) gene, which consists of 18 exons spanning 45 kb and codes for a precursor protein of 860 amino acids. Mutations in the LDLR gene lead to a reduced hepatic clearance of LDL as well as a high risk of coronary artery disease (CAD) and sudden cardiac death (SCD). Recently, LDLR transgenes have generated interest as potential therapeutic agents. However, LDLR packaging using a lentiviral vector (LVV) system pseudotyped with a vesicular stomatitis virus (VSV)-G envelope is not efficient. In this study, we modified the LVV system to improve transduction efficiency and investigated the LDLR regions responsible for transduction inhibition. Transduction efficiency of 293T cells with a 5'-LDLReGFP-3' fusion construct was only 1.55% compared to 42.32% for the eGFP construct. Moreover, co-expression of LDLR affected eGFP packaging. To determine the specific region of the LDLR protein responsible for packaging inhibition, we designed constructs with mutations or sequential deletions at the 3' and 5' ends of LDLR cDNA. All constructs except one without the ligand-binding domain (LBD) (pWoLBD-eGFP) resulted in low transduction efficiency, despite successful packaging of viral RNA in the VSV envelope, as confirmed through RT-PCR. When we evaluated a direct interaction between LDLR and the VSV envelope glycoprotein using MD simulation and protein-protein interactions, we uncovered Val119, Thr120, Thr67, and Thr118 as exposed residues in the LDLR receptor that interact with the VSV protein. Together, our results suggest that the LBD of LDLR interacts with the VSV-G protein during viral packaging, which significantly reduces transduction efficiency.
Assuntos
Glicoproteínas de Membrana/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Receptores de LDL/química , Proteínas do Envelope Viral/química , Sítios de Ligação , Linhagem Celular , Genes Reporter , Humanos , Glicoproteínas de Membrana/metabolismo , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão , Proteínas do Envelope Viral/metabolismoRESUMO
Mutations in the low-density lipoprotein receptor (LDLR), which cause familial hypercholesterolemia (FH), present a variable clinical FH phenotype. To date, over 1600 FH-causing mutations have been found worldwide. The aim of this study was to investigate the structure-function relationships of LDLR mutations by using homology modeling. Structural analysis of 36 missense mutations of known receptor activity (33 severe, 1 mild, and 2 non-pathogenic phenotypes) using sequence comparison and homology modeling was performed. Severe phenotypes had less than 2% to 32% of residual LDLR activity. Mild phenotypes had 76-92% of residual LDLR activity. Finally, non-pathogenic phenotypes had normal residual LDLR activity. Sequence comparisons showed that most of the severe phenotypes were located within the fully conserved residues of LDLR, while most of the mild and non-pathogenic phenotypes were located within the poorly conserved residues. Homology modeling demonstrated several phenomena for severe phenotypes: disruption of disulfide bond formation, disturbance of the calcium binding sites, and perturbation of LDLR hydrophobic conserved packing. In contrast, mild and non-pathogenic phenotypes did not disturb the critical region of LDLR. In addition, the root mean square deviation (RMSD) values of severe phenotype tended to be higher than the mild and non-pathogenic phenotypes, and the mean of solvent accessible surface area (ASA) of the residues in wild type structure for the severe phenotype was lower than mild and non-pathogenic phenotypes. These findings provide a better understanding in the structure-function relationships of LDLR mutations and may be useful in predicting FH severity based on future genotyping.