RESUMEN
Mammalian frataxin is a small mitochondrial protein involved in iron sulfur cluster assembly. Frataxin deficiency causes the neurodegenerative disease Friedreich's Ataxia. Valuable knowledge has been gained on the structural dynamics of frataxin, metal-ion-protein interactions, as well as on the effect of mutations on protein conformation, stability and internal motions. Additionally, laborious studies concerning the enzymatic reactions involved have allowed for understanding the capability of frataxin to modulate Fe-S cluster assembly function. Remarkably, frataxin biological function depends on its interaction with some proteins to form a supercomplex, among them NFS1 desulfurase and ISCU, the scaffolding protein. By combining multiple experimental tools including high resolution techniques like NMR and X-ray, but also SAXS, crosslinking and mass-spectrometry, it was possible to build a reliable model of the structure of the desulfurase supercomplex NFS1/ACP-ISD11/ISCU/frataxin. In this chapter, we explore these issues showing how the scientific view concerning frataxin structure-function relationships has evolved over the last years.
Asunto(s)
Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Ataxia de Friedreich/genética , Humanos , Proteínas de Unión a Hierro/genética , Dispersión del Ángulo Pequeño , Relación Estructura-Actividad , Difracción de Rayos X , FrataxinaRESUMEN
The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Apirasa/química , Apirasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Frío , Cristalografía por Rayos X , Estructura Terciaria de ProteínaRESUMEN
Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreich's Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (µs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the ß-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.
Asunto(s)
Proteínas de Unión a Hierro/química , Simulación de Dinámica Molecular , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Mutación , Estructura Secundaria de Proteína , Relación Estructura-Actividad , FrataxinaRESUMEN
Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.
Asunto(s)
Virus del Dengue/enzimología , ARN Helicasas/metabolismo , ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Sitios de Unión , Poli A/metabolismo , Unión Proteica , Multimerización de Proteína , ARN/química , ARN Helicasas/química , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Termodinámica , Proteínas no Estructurales Virales/químicaRESUMEN
Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a "hinge" region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t(1/2)â¼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 10(7) M(-1) s(-1), a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with K(D) = 1.2 × 10(-7) M is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be "locked" in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Antivirales/química , Especificidad de Anticuerpos , Papillomavirus Humano 16/química , Proteínas E7 de Papillomavirus/química , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Antivirales/inmunología , Epítopos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Humanos , Ratones , Resonancia Magnética Nuclear Biomolecular , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/inmunología , Estructura Secundaria de ProteínaRESUMEN
The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four ß-strands constituting the central ß-sheet of the αßα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Flavobacteriaceae/química , Secuencia de Aminoácidos , Animales , Regiones Antárticas , Proteínas Bacterianas/genética , Sitios de Unión , Calcio/metabolismo , Dicroismo Circular , Cristalografía por Rayos X , Metales/química , Metales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
A psychrotolerant marine bacterial strain, designated JUB59(T), was isolated from Antarctic surface seawater and classified as a new species of the genus Bizionia. Here, we present the first draft genome sequence for this genus, which suggests interesting features such as UV resistance, hydrolytic exoenzymes, and nitrogen metabolism.
Asunto(s)
Flavobacteriaceae/genética , Flavobacteriaceae/aislamiento & purificación , Genoma Bacteriano , Agua de Mar/microbiología , Regiones Antárticas , Secuencia de Bases , Flavobacteriaceae/clasificación , Datos de Secuencia Molecular , FilogeniaRESUMEN
Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionic strength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experiments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with a >or=2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed in excess of E2C. A discrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. The N-terminal domain of E7 interacts with E2C with a K(D) of 0.1 muM, where the stretch of residues 25-40 of E7, encompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement of the soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that the protein-protein interface involves the DNA binding helix of E2. The formation of complexes of different sizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that could regulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2 into E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Papillomavirus Humano 16/química , Papillomavirus Humano 16/genética , Humanos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Estructura Terciaria de Proteína , Integración Viral , Proteínas ras/antagonistas & inhibidores , Proteínas ras/metabolismoRESUMEN
Production of soluble recombinant proteins is crucial to the development of industry and basic research. However, the aggregation due to the incorrect folding of the nascent polypeptides is still a mayor bottleneck. Understanding the factors governing protein solubility is important to grasp the underlying mechanisms and improve the design of recombinant proteins. Here we show a quantitative study of the expression and solubility of a set of proteins from Bizionia argentinensis. Through the analysis of different features known to modulate protein production, we defined two parameters based on the %MinMax algorithm to compare codon usage clusters between the host and the target genes. We demonstrate that the absolute difference between all %MinMax frequencies of the host and the target gene is significantly negatively correlated with protein expression levels. But most importantly, a strong positive correlation between solubility and the degree of conservation of codons usage clusters is observed for two independent datasets. Moreover, we evince that this correlation is higher in codon usage clusters involved in less compact protein secondary structure regions. Our results provide important tools for protein design and support the notion that codon usage may dictate translation rate and modulate co-translational folding.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Flavobacteriaceae/genética , Biosíntesis de Proteínas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Codón , Escherichia coli/metabolismo , Flavobacteriaceae/metabolismo , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch expression in brains from patients at later stages of sporadic Alzheimer's disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/-)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/-) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aß) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/-) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and 1H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aß in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aß pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.
Asunto(s)
Enfermedad de Alzheimer/patología , Vasos Sanguíneos/patología , Glucosa/metabolismo , Hipocampo/metabolismo , Trastornos de la Memoria/patología , Receptores Notch/química , Receptores Notch/metabolismo , Memoria Espacial , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/fisiopatología , Animales , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Modelos Animales de Enfermedad , Vectores Genéticos/metabolismo , Células HEK293 , Hipocampo/patología , Hipocampo/fisiopatología , Humanos , Inflamación/patología , Lentivirus/genética , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/fisiopatología , Microvasos/patología , Dominios Proteicos , Espectroscopía de Protones por Resonancia Magnética , Ratas Transgénicas , Ratas WistarRESUMEN
The TPM domain constitutes a family of recently characterized protein domains that are present in most living organisms. Although some progress has been made in understanding the cellular role of TPM-containing proteins, the relationship between structure and function is not clear yet. We have recently solved the solution and crystal structure of one TPM domain (BA42) from the Antarctic bacterium Bizionia argentinensis. In this work, we demonstrate that BA42 has phosphoric-monoester hydrolase activity. The activity of BA42 is strictly dependent on the binding of divalent metals and retains nearly 70% of the maximum at 4 °C, a typical characteristic of cold-adapted enzymes. From HSQC, 15 N relaxation measurements, and molecular dynamics studies, we determine that the flexibility of the crossing loops was associated to the protein activity. Thermal unfolding experiments showed that the local increment in flexibility of Mg2+ -bound BA42, when compared with Ca2+ -bound BA42, is associated to a decrease in global protein stability. Finally, through mutagenesis experiments, we unambiguously demonstrate that the region comprising the metal-binding site participates in the catalytic mechanism. The results shown here contribute to the understanding of the relationship between structure and function of this new family of TPM domains providing important cues on the regulatory role of Mg2+ and Ca2+ and the molecular mechanism underlying enzyme activity at low temperatures.
Asunto(s)
Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Frío , Flavobacteriaceae/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Secuencia de Aminoácidos , Regiones Antárticas , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Estabilidad de Enzimas , Flavobacteriaceae/genética , Concentración de Iones de Hidrógeno , Cinética , Magnesio/metabolismo , Espectroscopía de Resonancia Magnética , Metales/metabolismo , Modelos Moleculares , Mutación , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
A gene encoding a protein classified as alanyl-tRNA synthetase (AlaRS) was found in the genome of the psychrophilic bacteria Bizionia argentinensis. The enzyme is constituted by three domains with an evolutionarily conserved modular arrangement: the N-terminal aminoacylation domain, the editing domain and the C-terminal domain (C-Ala). Herein we report the near complete NMR resonance assignment of the 122 amino acid C-Ala domain from B. argentinensis. The chemical shift data, reported for the first time for a C-Ala domain, constitute the basis for NMR structural studies aimed at elucidating the cold-adaptation mechanism of AlaRS.
Asunto(s)
Alanina-ARNt Ligasa/química , Flavobacteriaceae/enzimología , Resonancia Magnética Nuclear Biomolecular , Alanina-ARNt Ligasa/metabolismo , Estructura Terciaria de Proteína , ARN de Transferencia/metabolismoRESUMEN
BACKGROUND: Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric "Z-nucleus" after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 µM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a "conformation editing" mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route. CONCLUSION: We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions.
Asunto(s)
Papillomavirus Humano 16/química , Proteínas E7 de Papillomavirus/química , Multimerización de Proteína , Zinc/química , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Zinc/metabolismoRESUMEN
BA42 is a protein belonging to the psychrophilic bacteria Bizionia argentinensis sp. nov. Bioinformatics analysis showed that it presents significant sequence identity with a Pfam A family, DUF 477, found both in eukarya and eubacteria but of unknown function in all these organisms. Here, we report the NMR spectra assignment of this 145 amino acid protein. These data will allow performing NMR structural studies with the aim of using the three-dimensional structure as relevant information in order to determine the function of this family of proteins.
Asunto(s)
Proteínas Bacterianas/química , Flavobacteriaceae/metabolismo , Resonancia Magnética Nuclear Biomolecular , Protones , Secuencia de Aminoácidos , Isótopos de Carbono , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Estructura Secundaria de ProteínaRESUMEN
DNA tumor viruses ensure genome amplification by hijacking the cellular replication machinery and forcing infected cells to enter the S phase. The retinoblastoma (Rb) protein controls the G1/S checkpoint, and is targeted by several viral oncoproteins, among these the E7 protein from human papillomaviruses (HPVs). A quantitative investigation of the interaction mechanism between the HPV16 E7 protein and the RbAB domain in solution revealed that 90% of the binding energy is determined by the LxCxE motif, with an additional binding determinant (1.0 kcal.mol(-1)) located in the C-terminal domain of E7, establishing a dual-contact mode. The stoichiometry and subnanomolar affinity of E7 indicated that it can bind RbAB as a monomer. The low-risk HPV11 E7 protein bound 2.0 kcal.mol(-1) more weakly than the high-risk HPV16 and HPV18 type counterparts, but the modularity and binding mode were conserved. Phosphorylation at a conserved casein kinase II site in the natively unfolded N-terminal domain of E7 affected the local conformation by increasing the polyproline II content and stabilizing an extended conformation, which allowed for a tighter interaction with the Rb protein. Thus, the E7-RbAB interaction involves multiple motifs within the N-terminal domain of E7 and at least two conserved interaction surfaces in RbAB. We discussed a mechanistic model of the interaction of the Rb protein with a viral target in solution, integrated with structural data and the analysis of other cellular and viral proteins, which provided information about the balance of interactions involving the Rb protein and how these determine the progression into either the normal cell cycle or transformation.
Asunto(s)
Papillomavirus Humano 16 , Proteínas Oncogénicas Virales/metabolismo , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Quinasa de la Caseína II/genética , Secuencia Conservada , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteína de Retinoblastoma/genéticaRESUMEN
The E6 oncoproteins of high-risk HPV types 16 and 18 are involved in the development of cervical cancer. Besides its determinant role in carcinogenic progression, HPV E6 oncoprotein has also been instrumental in elucidating fundamental aspects of p53 function and its ubiquitin-proteasome degradation, with counterpart activities in various DNA tumor viruses. Establishing the conformational state and cellular distribution unequivocally for the endogenous protein in HPV-transformed cell lines derived from carcinomas is essential for understanding the underlying mechanism. Recombinant E6 from high-risk strains 16 and 18 folds into soluble oligomers of approximately 1.2 MDa, which are thermostable and display cooperative loss of tertiary and secondary structure upon chemical denaturation. Antibodies raised against these assemblies locate E6 evenly distributed in the cells. By depleting the polyclonal serum by immunoblocking with monomeric E6, the nuclei of Hela and CaSki cells become completely devoid of label, indicating that monomeric species are mainly localized in the nucleus and that both monomers and oligomers share epitopes. The monomeric species promote degradation of p53 by the proteasome, which correlates with the nuclear localization we describe. In contrast, the oligomeric E6 does not promote p53 degradation, in agreement with its cytoplasmic localization inferred from the immunoneutralization experiments. Our results indicate that the cytoplasmic species contain conformational epitopes that may arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets, those involving PDZ binding domains, which requires further investigation.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Oncogénicas Virales/química , Proteínas Represoras/química , Secuencia de Bases , Línea Celular Transformada , Transformación Celular Neoplásica , Transformación Celular Viral , ADN Viral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Femenino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidad , Papillomavirus Humano 16/fisiología , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidad , Papillomavirus Humano 18/fisiología , Humanos , Modelos Biológicos , Complejos Multiproteicos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/fisiología , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/etiología , Neoplasias del Cuello Uterino/virologíaRESUMEN
E7 oncoprotein is the major transforming activity in human papillomavirus and shares sequence and functional properties with adenovirus E1A and SV40 T-antigen, in particular by targeting the pRb tumor suppressor. HPV 16 E7 forms spherical oligomers that display chaperone activity in thermal denaturation and chemical refolding assays of two model polypeptide substrates, citrate synthase and luciferase, and it does so at substoichiometric concentrations. We show that the E7 chaperone can stably bind model polypeptides and hold them in a state with significant tertiary structure, but does not bind the fully native proteins. The E7 oligomers bind native in vitro translated pRb without the requirement of it being unfolded, since the N-terminal domain of E7 containing the LXCXE binding motif is exposed. The N-terminal domain of E7 can interfere with pRb binding but not with the chaperone activity, which requires the C-terminal domain, as in most reported E7 activities. The ability to bind up to approximately 72 molecules of pRb by the oligomeric E7 form could be important either for sequestering pRb from Rb-E2F complexes or for targeting it for proteasome degradation. Thus, both the dimeric and oligomeric chaperone forms of E7 can bind Rb and various potential targets. We do not know at present if the chaperone activity of E7 plays an essential role in the viral life cycle; however, a chaperone activity may explain the large number of cellular targets reported for this oncoprotein.
Asunto(s)
Chaperonas Moleculares/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/enzimología , Cinética , Modelos Moleculares , Chaperonas Moleculares/genética , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus , Conformación Proteica , Desnaturalización ProteicaRESUMEN
Despite the fact that E7 is a major transforming oncoprotein in papillomavirus, its structure and precise molecular mechanism of action remain puzzling to date. E7 proteins share sequence homology and proteasome targeting properties of tumor suppressors with adenovirus E1A and SV40 T antigen, two other paradigmatic oncoproteins from DNA tumor viruses. High-risk HPV16 E7, a nonglobular dimer with some properties of intrinsically disordered proteins, is capable of undergoing pH-dependent conformational transitions that expose hydrophobic surfaces to the solvent. We found that treatment with a chelating agent produced a protein that can readily assemble into homogeneous spherical particles with an average molecular mass of 790 kDa and a diameter of 50 nm, as determined from dynamic light scattering and electron microscopy. The protein undergoes a substantial conformational transition from coil to beta-sheet structure, with concomitant consolidation of tertiary structure as judged by circular dichroism and fluorescence. The assembly process is very slow, in agreement with a substantial energy barrier caused by structural rearrangements. The resulting particles are highly stable, cooperatively folded, and capable of binding both Congo Red and thioflavin T, reporters of repetitive beta-sheet structures similar to those found in amyloids, although no fibrillar or insoluble material was observed under our experimental conditions.