Assuntos
5-Aminolevulinato Sintetase/genética , Predisposição Genética para Doença , Proteína da Hemocromatose/genética , Sobrecarga de Ferro/diagnóstico , Sobrecarga de Ferro/etiologia , Mutação , 5-Aminolevulinato Sintetase/química , Adulto , Biomarcadores , Biópsia , Feminino , Estudos de Associação Genética , Proteína da Hemocromatose/química , Humanos , Perda de Heterozigosidade , Imageamento por Ressonância Magnética , Linhagem , Fenótipo , Índice de Gravidade de Doença , Relação Estrutura-Atividade , Avaliação de SintomasRESUMO
Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.
Assuntos
Antígenos CD/química , Apoferritinas/química , Proteínas de Protozoários/química , Receptores da Transferrina/química , Receptores Virais/química , Transferrina/química , Proteínas do Envelope Viral/química , Antígenos CD/genética , Antígenos CD/metabolismo , Apoferritinas/genética , Apoferritinas/metabolismo , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Proteína da Hemocromatose/química , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Humanos , Plasmodium vivax/genética , Plasmodium vivax/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transferrina/genética , Transferrina/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Hereditary hemochromatosis is an iron overburden condition, which is mainly governed by hereditary hemochromatosis factor E (HFE), a member of major histocompatibility complex class I. To understand the effect of pH on the structure and stability of HFE, we have cloned, expressed, and purified the HFE in the bacterial system and performed circular dichroism, fluorescence, and absorbance measurements at a wide pH range (pH 3.0-11.0). We found that HFE remains stable in the pH range 7.5-11.0 and gets completely acid denatured at low pH values. In this work, we also analyzed the contribution of salt bridges to the stability of HFE. We further performed molecular dynamics simulations for 80 ns at different pH values. An excellent agreement was observed between results from biophysical and MD simulation studies. At lower pH, HFE undergoes denaturation and may be driven toward a degradation pathway, such as ubiquitination. Hence, HFE is not available to bind again with transferrin receptor1 to negatively regulate iron homeostasis. Further we postulated that, might be low pH of cancerous cells helps them to meet their high iron requirement.
Assuntos
Proteína da Hemocromatose/química , Simulação de Dinâmica Molecular , Motivos de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Desnaturação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Hereditary hemochromatosis factor E (HFE) is a type 1 transmembrane protein, and acts as a negative regulator of iron-uptake. The equilibrium unfolding and conformational stability of the HFE protein was examined in the presence of urea. The folding and unfolding transitions were monitored with the help of circular dichroism (CD), intrinsic fluorescence and absorption spectroscopy. Analysis of transition curves revealed that the folding of HFE is not a two-state process. However, it involved stable intermediates. Transition curves (plot of fluorescence (F346) and CD signal at 222nm (θ222) versus [Urea], the molar urea concentration) revealed a biphasic transition with midpoint (Cm) values at 2.88M and 4.95M urea. Whereas, absorption analysis shows one two-state transition centered at 2.96M. To estimate the protein stability, denaturation curves were analyzed for Gibbs free energy change in the absence of urea (ΔGD(0)) associated with the equilibrium of denaturation exist between native stateâdenatured state. The intermediate state was further characterized by hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS-binding). For seeing the effect of urea on the structure and dynamics of HFE, molecular dynamics simulation for 60ns was also performed. A clear correspondence was established between the in vitro and in silico studies.
Assuntos
Proteína da Hemocromatose/química , Desdobramento de Proteína/efeitos dos fármacos , Ureia/farmacologia , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacosRESUMO
Hemochromatosis factor E (HFE) is a member of class I MHC family and plays a significant role in the iron homeostasis. Denaturation of HFE induced by guanidinium chloride (GdmCl) was measured by monitoring changes in [θ]222 (mean residue ellipticity at 222 nm), intrinsic fluorescence emission intensity at 346 nm (F346 ) and the difference absorption coefficient at 287 nm (Δε287) at pH 8.0 and 25°C. Coincidence of denaturation curves of these optical properties suggests that GdmCl-induced denaturation (native (N) state â denatured (D) state) is a two-state process. The GdmCl-induced denaturation was found reversible in the entire concentration range of the denaturant. All denaturation curves were analyzed for ΔGD0, Gibbs free energy change associated with the denaturation equilibrium (N state â D state) in the absence of GdmCl, which is a measure of HFE stability. We further performed molecular dynamics simulation for 40 ns to see the effect of GdmCl on the structural stability of HFE. A well defined correlation was established between in vitro and in silico studies.
