PubMed Health⌕ Search

Biomedical subjects

Gonzalo de Prat-Gay

Publications and source records attributed to Gonzalo de Prat-Gay.

16 recordsLinked to original sources
PubMed2007

High-risk HPV E6 oncoproteins assemble into large oligomers that allow localization of endogenous species in prototypic HPV-transformed cell lines.

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.

Base Sequence↗
PubMed2006

Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2.

We have obtained a monoclonal antibody (ED15) against the C-terminal DNA-binding domain of the high-risk human papillomavirus strain-16 E2 protein that strongly interferes with its DNA-binding activity. We here characterize the recognition mechanism of this antibody and find that the ED15-E2 interaction has a strong electrostatic component, which correlates with the high proportion of acidic residues found in the antibody combining site. Further circular dichroism experiments in the presence of phosphate show that, in addition to electrostatic screening of key potential interactions, ionic strength affects the conformation of the epitope. In addition, the interaction is strongly modulated by pH, which correlates with the local flexibility of the epitope rather than the presence of pH sensitive residues at the interface. Noticeably, this finding is well correlated with the strong entropic component of the interaction. Site directed mutagenesis indicates that the ED15 epitope involves at least part of the DNA-binding helix of E2, explaining the mAb inhibitory activity. At physiological salt concentrations, the equilibrium dissociation constant of the E2-ED15 interaction is 10(-7) M and the association rate is 10(4) M-1 s-1, at least 1 order of magnitude slower than those generally reported in the most extensively described "nonflexible" antibody-protein interactions, indicating the presence of a slow conformational rearrangement on the antigen as the rate-limiting step. The crucial role of antigen flexibility in antibody-protein recognition is discussed.

Amino Acid Sequence↗
PubMed2006

Mechanism of DNA recognition at a viral replication origin.

Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1452-641, to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol-1, with 11.9 kcal mol-1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1452-641.DNA complex is accompanied by a heat capacity change of -1.22 kcal mol-1 K-1, a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1.DNA complex conformers are in slow equilibrium; one dissociates slowly (t1/2 approximately 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1/2 approximately 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6x10(8) m-1 s-1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of approximately 2.0 s-1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.

Animals↗
PubMed2006

Structural and thermodynamic basis for the enhanced transcriptional control by the human papillomavirus strain-16 E2 protein.

Strain 16 of the human papillomavirus is responsible for the largest number of cases of cervical cancers linked to this virus, and the E2 protein is the transcriptional regulator of all viral genes. We present the first structure for the DNA binding domain of HPV16 E2 bound to DNA, and in particular, to a natural cognate sequence. The NMR structure of the protein backbone reveals that the overall conformation remains virtually unchanged, and chemical shift analysis of the protein bound to a shorter DNA duplex uncovered a contact out of the minimal E2 DNA binding site, made by lysine 349. This contact was confirmed by titration calorimetry and mutagenesis, with a contribution of 1.0 kcal mol(-)(1) to binding energy. HPV16 E2 has the highest DNA binding affinity and exerts a strict transcriptional control, translated into the repression of the E6 and E7 oncogenes. These novel features provide the structural and thermodynamic basis for this tight transcriptional control, the loss of which correlates with carcinogenesis.

Amino Acid Sequence↗
PubMed2006

Chaperone holdase activity of human papillomavirus E7 oncoprotein.

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.

Kinetics↗
PubMed2006

Molecular basis for phosphorylation-dependent, PEST-mediated protein turnover.

Proteasomal-mediated rapid turnover of proteins is often modulated by phosphorylation of PEST sequences. The E2 protein from papillomavirus participates in gene transcription, DNA replication, and episomal genome maintenance. Phosphorylation of a PEST sequence located in a flexible region accelerates its degradation. NMR analysis of a 29 amino acid peptide fragment derived from this region shows pH-dependent polyproline II and alpha helix structures, connected by a turn. Phosphorylation, in particular that at serine 301, disrupts the overall structure, and point mutations have either stabilizing or destabilizing effects. There is an excellent correlation between the thermodynamic stability of different peptides and the half-life of E2 proteins containing the same mutations in vivo. The structure around the PEST region appears to have evolved a marginal stability that is finely tunable by phosphorylation. Thus, conformational stability, rather than recognition of a phosphate modification, modulates the degradation of this PEST sequence by the proteasome machinery.

Amino Acid Motifs↗
PubMed2005

The folding mechanism of a dimeric beta-barrel domain.

The dimeric beta-barrel domain is an unusual topology, shared only by two viral origin binding proteins, where secondary, tertiary and quaternary structure are coupled, and where the dimerization interface is composed of two four-stranded half-beta-barrels. The folding of the DNA binding domain of the E2 transcriptional regulator from human papillomavirus, strain-16, takes place through a stable and compact monomeric intermediate, with 31% the stability of the folded dimeric domain. Double jump multiple wavelength experiments allowed the reconstruction of the fluorescence spectrum of the monomeric intermediate at 100 milliseconds, indicating that tryptophan residues, otherwise buried in the folded state, are accessible to the solvent. Burial of surface area as well as differential behavior to ionic strength and pH with respect to the native ground state, plus the impossibility of having over 2500 A2 of surface area of the half-barrel exposed to the solvent, indicates that the formation of a non-native compact tertiary structure precedes the assembly of native quaternary structure. The monomeric intermediate can dimerize, albeit with a weaker affinity (approximately 1 microM), to yield a non-native dimeric intermediate, which rearranges to the native dimer through a parallel folding channel, with a unimolecular rate-limiting step. Folding pathways from either acid or urea unfolded states are identical, making the folding model robust. Unfolding takes place through a major phase accounting for apparently all the secondary structure change, with identical rate constant to that of the fluorescence unfolding experiment. In contrast to the folding direction, no unfolding intermediate was found.

Dimerization↗
PubMed2005

Insulin-degrading enzyme degrades amyloid peptides associated with British and Danish familial dementia.

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant disorders characterized by cerebrovascular and parenchymal amyloid deposition and neurofibrillary degeneration. In both conditions, the genetic defects cause the loss of the normal stop codon in the precursor BRI, generating novel 34-residue peptides named ABri and ADan in FBD and FDD, respectively. ABri and ADan show a strong tendency to aggregate into non-fibrillar and fibrillar structures at neutral pH and this property seems to be directly related to neurotoxicity. Here we report that a recombinant insulin-degrading enzyme (rIDE) was capable of degrading monomeric ABri and ADan in vitro more efficiently than oligomeric species. These peptides showed high beta-structure content and were more resistant to proteolysis as compared to the BRI wild-type product of 23 amino acids. Specific sites of cleavage within the C-terminal pathogenic extensions raise the possibility that proteolysis of monomeric soluble precursors by IDE may delay ABri and ADan aggregation in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Free energy contributions to direct readout of a DNA sequence.

The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.

Amino Acid Sequence↗
PubMed2004

The endoplasmic reticulum glucosyltransferase recognizes nearly native glycoprotein folding intermediates.

The UDP-Glc:glycoprotein glucosyltransferase (GT), a key player in the endoplasmic reticulum (ER) quality control of glycoprotein folding, only glucosylates glycoproteins displaying non-native conformations. To determine whether GT recognizes folding intermediates or irreparably misfolded species with nearly native structures, we generated and tested as GT substrates neoglycoprotein fragments derived from chymotrypsin inhibitor 2 (GCI2) bearing from 53 to 64 (full-length) amino acids. Fragment conformations mimicked the last stage-folding structures adopted by a glycoprotein entering the ER lumen. GT catalytic efficiency (V(max)/K(m)) remained constant from GCI2-(1-53) to GCI2-(1-58) and then steadily declined to reach a minimal value with GCI2-(1-64). The same parameter showed a direct hyperbolic relationship with solvent accessibility of the single Trp residue but only in fragments exposing hydrophobic amino acid patches. Mutations introduced (GCI2-(1-63)V63S and GCI2-(1-64)V63S) produced slight structural destabilizations but increased GT catalytic efficiency. This parameter presented an inverse exponential relationship with the free energy of unfolding of canonical and mutant fragments. Moreover, the catalytic efficiency showed a linear relationship with the fraction of unfolded species in water. It was concluded that the GT-derived quality control may be operative with nearly native conformers and that no alternative ER-retaining mechanisms are required when glycoproteins approach their proper folding.

Animals↗
PubMed2004

The HPV16 E7 viral oncoprotein self-assembles into defined spherical oligomers.

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.

Benzothiazoles↗
PubMed2004

Casein Kinase II phosphorylation-induced conformational switch triggers degradation of the papillomavirus E2 protein.

The major phosphorylation sites of the bovine papillomavirus E2 transactivator protein are two serine residues, 298 and 301, that are located in a flexible hinge region between the DNA binding and transactivation domains. Phosphorylation of serine residue 301 promotes ubiquitination and rapid degradation of the E2 protein by the proteasome pathway. To understand the mechanism through which phosphorylation regulates the intracellular levels of this unique papillomavirus regulatory protein, we have carried out an extensive mutational analysis of the region surrounding the phosphorylation sites of the E2 protein. Our results indicate that casein kinase II phosphorylates serine 301. However, phosphorylation of serine 301 is not a sufficient recognition motif for proteasomal degradation; other residues that directly surround the phosphorylation sites are crucial for E2 degradation. The phenotypes of E2 proteins mutated in this region indicate that phosphorylation of serine 301 induces a conformational change that leads to degradation of the E2 protein. In support of this model, circular dichroism studies of the conformational tendencies of peptides from this region indicate that phosphorylation at position 301 decreases the local thermodynamic stability of this region. Thus, this region appears to have evolved to display a marginal local thermodynamic stability that can be regulated by phosphorylation, leading to targeted degradation of the E2 protein.

Amino Acid Motifs↗
PubMed2003

A protein-DNA binding mechanism proceeds through multi-state or two-state parallel pathways.

The DNA-binding mechanism of the dimeric C-terminal domain of the papillomavirus E2 protein with its specific DNA target was investigated and shown to proceed through two parallel pathways. A sequential multi-step reaction is initiated by the diffusion-controlled formation of an encounter complex, with no evidence of base sequence discrimination capacity. Following a substantial conformational rearrangement of the protein, a solvent exclusion step leading to the formation of a final protein-DNA complex was identified. This last step involves the largest burial of surface area from the interface and involves the consolidation of the direct readout of the DNA bases. Double-jump stopped-flow experiments allowed us to characterize the sequence of events and demonstrated that a fast-formed consolidated complex can take place through a parallel route. We present the simplest model for the overall mechanism with a description of all the intermediate species in energetic terms.

Base Sequence↗
PubMed2003

Specific antibody-DNA interaction: a novel strategy for tight DNA recognition.

Anti-double-stranded DNA monoclonal antibodies against a viral transcriptional regulatory site are capable of discriminating single-base replacements with affinities of 1 x 10(-)(9) M, which were optimized for the length of the duplex used as the immunogen. Their affinity for DNA duplexes of increasing length is lower, but reaches a plateau at 2 x 10(-)(8) M, still a fairly high affinity compared to those of most known natural anti-DNA antibodies. The ability of the antibodies to bind to a 166 bp DNA fragment containing the specific sequence strongly suggests that these have the potential of binding the specific sequence within larger genomic DNA fragments. Electrostatic interactions do not play a significant role, the opposite of what is observed in natural DNA binding interfaces. In addition, the insensitivity of the antibody-DNA interaction to solute effects is indicative of a marginal participation of water molecules at the interface compared to the level of participation at the natural E2-DNA interface. Spectroscopic evidence of base unstacking strongly suggests substantial denaturation of antibody-bound DNA, in agreement with thermodynamic results that show an unusual positive heat capacity change, which could be explained at least in part by the exposure of DNA bases upon binding. Lower local DNA stability cooperates with sequence recognition in producing the highest binding affinity. A slow rate of antibody-DNA association indicates an energy barrier imposed by conformational rearrangements, as opposed to an electrostatically assisted diffusion-controlled collision in the E2 DNA binding domain. While the E2-DNA interaction takes place through a typical direct readout mechanism, the anti-double-stranded DNA monoclonal antibody-DNA interaction could be viewed as a distinctive case of indirect readout with a significant distortion in the DNA conformation. However, the precise mechanism with which the DNA bases are accommodated in the antibody combining site will require structural analysis at atomic resolution. These results constitute a first stage for unveiling the unusual molecular recognition mechanism of a specific DNA sequence by antibodies. This mechanism could represent the strategy with which the immune system tightly and specifically recognizes a DNA antigen.

Animals↗
PubMed2003

Antibody response to a viral transcriptional regulator.

The E2 transcriptional activator of the human papillomavirus regulates the expression of most viral transcripts. Its binding to specific target DNA sequences involves large conformational changes in the interacting macromolecules. The high stability of the E2:DNA complex prompted us to analyze the role of macromolecular interactions and adjuvant emulsions in the appearance of conformation-specific antibodies. We demonstrate that immunization with free or DNA-complexed E2 emulsified in an oil-in-water adjuvant elicits a humoral response shifted to the recognition of discontinuous epitopes. Epitope mapping and functional analysis of the generated anti-E2 mAbs reveals that two separate antibodies populations can be obtained: those able to form a stable ternary complex with protein and DNA, and those which recognize the DNA-binding surface of the transcription factor, interfering with E2 binding to DNA.

Adjuvants, Immunologic↗