NGOME-Lite: Proteome-wide prediction of spontaneous protein deamidation highlights differences between taxa.

Asparagines in proteins deamidate spontaneously, which changes the chemical structure of a protein and often affects its function. Current prediction algorithms for asparagine deamidation require a structure as an input or are too slow to be applied at a proteomic scale. We present NGOME-Lite, a new version of our sequence-based predictor for spontaneous asparagine deamidation that is faster by over two orders of magnitude at a similar degree of accuracy. The algorithm takes into account intrinsic sequence propensities and slowing down of deamidation by local structure. NGOME-Lite can run in a proteomic analysis mode that provides the half-time of the intact form of each protein, predicted by taking into account sequence propensities and structural protection or sequence propensities only, and a structure protection factor. The detailed analysis mode also provides graphical output for all Asn residues in the query sequence. We applied NGOME-Lite to over 257,000 sequences in 38 proteomes and found that different taxa differ in their predicted deamidation dynamics. Spontaneous protein deamidation is faster in Eukarya than in Bacteria because of a higher degree of structural protection in the latter. Predicted protein deamidation half-lifes correlate with protein turnover in human, mouse, rat, C. elegans and budding yeast but not in two plants and two bacteria. NGOME-Lite is implemented in a docker container available at https://ngome.proteinphysiologylab.org.

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif.

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.

Rapid and cost-effective process based on insect larvae for scale-up production of SARS-COV-2 spike protein for serological COVID-19 testing.

Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this study, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.

Topology Dictates Evolution of Regulatory Cysteines in a Family of Viral Oncoproteins.

Redox regulation in biology is largely operated by cysteine chemistry in response to a variety of cell environmental and intracellular stimuli. The high chemical reactivity of cysteines determines their conservation in functional roles, but their presence can also result in harmful oxidation limiting their general use by proteins. Papillomaviruses constitute a unique system for studying protein sequence evolution since there are hundreds of anciently evolved stable genomes. E7, the viral transforming factor, is a dimeric, cysteine-rich oncoprotein that shows both conserved structural and variable regulatory cysteines constituting an excellent model for uncovering the mechanism that drives the acquisition of redox-sensitive groups. By analyzing over 300 E7 sequences, we found that although noncanonical cysteines show no obvious sequence conservation pattern, they are nonrandomly distributed based on topological constrains. Regulatory residues are strictly excluded from six positions stabilizing the hydrophobic core while they are enriched in key positions located at the dimerization interface or around the Zn+2 ion. Oxidation of regulatory cysteines is linked to dimer dissociation, acting as a reversible redox-sensing mechanism that triggers a conformational switch. Based on comparative sequence analysis, molecular dynamics simulations and biophysical analysis, we propose a model in which the occurrence of cysteine-rich positions is dictated by topological constrains, providing an explanation to why a degenerate pattern of cysteines can be achieved in a family of homologs. Thus, topological principles should enable the possibility to identify hidden regulatory cysteines that are not accurately detected using sequence based methodology.

Conformational Isomerization Involving Conserved Proline Residues Modulates Oligomerization of the NS1 Interferon Response Inhibitor from the Syncytial Respiratory Virus.

Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main ß-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes.

Hidden Structural Codes in Protein Intrinsic Disorder.

Intrinsic disorder is a major structural category in biology, accounting for more than 30% of coding regions across the domains of life, yet consists of conformational ensembles in equilibrium, a major challenge in protein chemistry. Anciently evolved papillomavirus genomes constitute an unparalleled case for sequence to structure-function correlation in cases in which there are no folded structures. E7, the major transforming oncoprotein of human papillomaviruses, is a paradigmatic example among the intrinsically disordered proteins. Analysis of a large number of sequences of the same viral protein allowed for the identification of a handful of residues with absolute conservation, scattered along the sequence of its N-terminal intrinsically disordered domain, which intriguingly are mostly leucine residues. Mutation of these led to a pronounced increase in both α-helix and ß-sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics, and uncovers the existence of local structural elements that oppose canonical folding. These folding relays suggest the existence of yet undefined hidden structural codes behind intrinsic disorder in this model protein. Thus, evolution pinpoints conformational hot spots that could have not been identified by direct experimental methods for analyzing or perturbing the equilibrium of an intrinsically disordered protein ensemble.

Amino Acid metabolism conflicts with protein diversity.

The 20 protein-coding amino acids are found in proteomes with different relative abundances. The most abundant amino acid, leucine, is nearly an order of magnitude more prevalent than the least abundant amino acid, cysteine. Amino acid metabolic costs differ similarly, constraining their incorporation into proteins. On the other hand, a diverse set of protein sequences is necessary to build functional proteomes. Here, we present a simple model for a cost-diversity trade-off postulating that natural proteomes minimize amino acid metabolic flux while maximizing sequence entropy. The model explains the relative abundances of amino acids across a diverse set of proteomes. We found that the data are remarkably well explained when the cost function accounts for amino acid chemical decay. More than 100 organisms reach comparable solutions to the trade-off by different combinations of proteome cost and sequence diversity. Quantifying the interplay between proteome size and entropy shows that proteomes can get optimally large and diverse.

Cysteine-rich positions outside the structural zinc motif of human papillomavirus E7 provide conformational modulation and suggest functional redox roles.

The E7 protein from high-risk human papillomavirus is essential for cell transformation in cervical, oropharyngeal, and other HPV-related cancers, mainly through the inactivation of the retinoblastoma (Rb) tumor suppressor. Its high cysteine content (~7%) and the observation that HPV-transformed cells are under oxidative stress prompted us to investigate the redox properties of the HPV16 E7 protein under biologically compatible oxidative conditions. The seven cysteines in HPV16 E7 remain reduced in conditions resembling the basal reduced state of a cell. However, under oxidative stress, a stable disulfide bridge forms between cysteines 59 and 68. Residue 59 has a protective effect on the other cysteines, and its mutation leads to an overall increase in the oxidation propensity of E7, including cysteine 24 central to the Rb binding motif. Gluthationylation of Cys 24 abolishes Rb binding, which is reversibly recovered upon reduction. Cysteines 59 and 68 are located 18.6 Å apart, and the formation of the disulfide bridge leads to a large structural rearrangement while retaining strong Zn association. These conformational and covalent changes are fully reversible upon restoration of the reductive environment. In addition, this is the first evidence of an interaction between the N-terminal intrinsically disordered and the C-terminal globular domains, known to be highly and separately conserved among human papillomaviruses. The significant conservation of such noncanonical cysteines in HPV E7 proteins leads us to propose a functional redox activity. Such an activity adds to the previously discovered chaperone activity of E7 and supports the picture of a moonlighting pathological role of this paradigmatic viral oncoprotein.

Long-lasting immunoprotective and therapeutic effects of a hyperstable E7 oligomer based vaccine in a murine human papillomavirus tumor model.

Cervical cancer and many other anogenital and oropharyngeal carcinomas are strongly associated with high-risk human papillomavirus (HPV) persistent infections. HPV E7 oncoprotein is the major viral transforming factor, emerging as a natural candidate for immunotherapy, since it is constitutively expressed in HPV-induced cancer cells. We have previously shown that E7 can self-assemble into soluble and homogeneous spherical oligomers, named E7 soluble oligomers (E7SOs). These are highly resistant to thermal denaturation, providing an additional advantage given the demand for highly stable vaccine formulations. Here, we present a new chemically stabilized form of the E7SOs (E7SOx) and analyzed its effect in a murine HPV-tumor model. Vaccination of female mice with low doses of E7SOx combined with a CpG-rich oligonucleotide (ODN) as adjuvant elicits a strong long-lasting protection against E7-expressing tumor cells, preventing tumor outgrowth after rechallenge 90-days later. Therapeutic experiments showed that E7SOx/ODN vaccination significantly delays tumor growth and extends the time of survival of the treated mice in a dose-dependent manner. These proof-of-principle preclinical experiments denote the potential applicability of our E7SOx-based vaccine to the treatment of cervical cancer and other mucosal HPV-related neoplastic lesions. In addition to thermal, chemical and proteolysis stability, the combined recombinant and chemical modification nature of the E7SOx vaccine candidate, results in low-cost, of particular interest in developing countries, where most of the cervical cancer cases occur and the most affected population is at reproductive age.

RapD Is a Multimeric Calcium-Binding Protein That Interacts With the Rhizobium leguminosarum Biofilm Exopolysaccharide, Influencing the Polymer Lengths.

Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix's properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.

Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.

Novel bridge multi-species ELISA for detection of SARS-CoV-2 antibodies.

Considering the course of the current SARS-CoV-2 pandemic, it is important to have serological tests for monitoring humoral immune response against SARS-CoV-2 infection and vaccination. Herein we describe a novel bridge enzyme-linked immunosorbent assay (b-ELISA) for SARS-CoV-2 antibodies detection in human and other species, employing recombinant Spike protein as a unique antigen, which is produced at high scale in insect larvae. METHODS: Eighty two human control sera/plasmas and 169 COVID-19 patients' sera/plasmas, confirmed by rRT-PCR, were analyzed by the b-ELISA assay. In addition, a total of 27 animal sera (5 horses, 13 rats, 2 cats and 7 dogs) were employed in order to evaluate the b-ELISA in other animal species. RESULTS: Out of the 169 patient samples, 129 were positive for IgG anti-SARS-CoV-2 and 40 were negative when they were tested by ELISA COVIDAR® IgG. When a cut-off value of 5.0 SDs was established, 124 out of the 129 COVID-19 positive samples were also positive by our developed b-ELISA (sensitivity: 96.12%). Moreover, the test was able to evaluate the humoral immune response in animal models and also detected as positive a naturally infected cat and two dogs with symptoms, whose owners had suffered the COVID-19 disease. CONCLUSION: The obtained results demonstrate that the method developed herein is versatile, as it is able to detect antibodies against SARS-CoV-2 in different animal species without the need to perform and optimize a new assay for each species.

E6*, the 50 amino acid product of the most abundant spliced transcript of the e6 oncoprotein in high-risk human papillomavirus, is a promiscuous folder and binder.

High-risk human papillomavirus E6 participates in tumorigenic progression, mainly by its ability to promote p53 degradation. HPV transcripts show a complex splicing pattern, where E6* is the most abundant transcript in high-risk HPV types, comprising the first 50 amino acids of E6. No structural or biochemical information of this polypeptide, which contains half of the first zinc binding motif of E6, is available, due to the difficulty to acquire a compact monomeric fold in such a small polypeptide. We show that HPV16-E6* can fold into either α-helix or ß-sheet large oligomers at pH 7.5 and 5.0, respectively, in the absence of zinc. The ß-sheet oligomers are highly stable and unaffected by the presence of zinc, while the α-helix oligomers tend to rapidly form aggregates, prevented by the presence of the metal. Two E6* molecules bind per atom of zinc, suggesting a tetrahedral, high-affinity arrangement (K(D) < 10(-12) M), which results in a zinc-mediated E6* dimer with significant secondary structure. Endogenous E6 oligomers were previously found in the cytosol of high-risk HPV transformed cell lines, and we propose that the oligomerization determinant resides within E6*. E6* effects were reported to counteract those of E6 in cells, and the ratio between these two species modulates p53 degradation and other apoptosis-dependent signaling cascades. A residue of an evolved splicing event related to regulation of oncogene expression in HPV or a splicing event resulting from the selection of a small deleterious viral polypeptide, the abundant existence of E6* with a "chameleon" nature correlates with target plasticity, and its fate is linked to a balance between protein levels, zinc availability, redox potential, and oligomerization. In addition, the results presented here have strong implications for zinc binding sites in nascent polypeptides. This evolved promiscuous folder speaks of effect rather than function of a viral product that, when highly increased, can directly or indirectly affect various cellular processes leading to cell deregulation and tumorigenesis.

The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins.

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.

Cytosolic accumulation of HPV16 E7 oligomers supports different transformation routes for the prototypic viral oncoprotein: the amyloid-cancer connection.

E7 is the major transforming activity in human papillomaviruses, a causal agent for cervical cancer. HPV16 E7 is a small protein with a natively unfolded domain for which dozens of specific cellular targets were described, and represents a prototypical oncoprotein among small DNA tumor viruses. The protein can form spherical oligomers with amyloid-like properties and chaperone activity. Conformation specific antibodies locate endogenous oligomeric E7 species in the cytosol of 3 model cell lines, strongly co-localizing with amyloid structures and dimeric E7 localizes to the nucleus. The cytosolic oligomeric E7 appear as the most abundant species in all cell systems tested. We show that nuclear E7 levels are replenished dynamically from the cytosolic pool and do not result from protein synthesis. Our results suggest that long-term events related to de-repression of E7 would cause accumulation of excess E7 into oligomeric species in the cytosol. These, together with the known target promiscuity of E7, may allow interactions with many of the non-pRb dependent targets described. This hypothesis is further supported by the detection of E7 oligomers in the cytosol of cancerous cells from tissue biopsies.

SPARC modulates the proliferation of stromal but not melanoma cells unless endogenous SPARC expression is downregulated.

Cell interaction with the extracellular matrix (ECM) has profound influence in cancer progression. The secreted protein, acidic and rich in cysteine (SPARC) a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. This apparent paradox might result either from the biochemical properties of the different SPARC sources or from differential responses of malignant and stromal cells to SPARC. To test these hypotheses, we purified SPARC secreted by melanoma cells (hMel-SPARC) and compared its activity with different recombinant SPARC preparations, including a new one produced in insect cells. All 5 SPARC species were effective in inhibiting bovine aortic endothelial cell proliferation, adhesion and migration. We then used the melanoma-derived protein to assess SPARC effect on additional cell types. hMel-SPARC greatly impaired the proliferation of both normal and transformed human endothelial cells and exerted a moderate biphasic effect on human fetal fibroblasts proliferation, irrespective of their endogenous SPARC levels. However, SPARC had no effect on the proliferation of several human cancer cell lines regardless of their endogenous levels of SPARC expression. Importantly, downregulation of SPARC levels in melanoma cells using either an antisense RNA or a shRNA against SPARC sensitized them to hMel-SPARC addition in proliferation and migration assays, suggesting that malignant cells developed a SPARC-resistance mechanism. This was not a general resistance to growth suppressing agents, as melanoma cells with restricted SPARC expression were more resistant to chemotherapeutic agents. Thus, malignant cells expressing or not expressing SPARC developed alternative mechanisms that, in contrary to stromal cells, rendered them SPARC-insensitive.

Degenerate cysteine patterns mediate two redox sensing mechanisms in the papillomavirus E7 oncoprotein.

Infection with oncogenic human papillomavirus induces deregulation of cellular redox homeostasis. Virus replication and papillomavirus-induced cell transformation require persistent expression of viral oncoproteins E7 and E6 that must retain their functionality in a persistent oxidative environment. Here, we dissected the molecular mechanisms by which E7 oncoprotein can sense and manage the potentially harmful oxidative environment of the papillomavirus-infected cell. The carboxy terminal domain of E7 protein from most of the 79 papillomavirus viral types of alpha genus, which encloses all the tumorigenic viral types, is a cysteine rich domain that contains two classes of cysteines: strictly conserved low reactive Zn+2 binding and degenerate reactive cysteine residues that can sense reactive oxygen species (ROS). Based on experimental data obtained from E7 proteins from the prototypical viral types 16, 18 and 11, we identified a couple of low pKa nucleophilic cysteines that can form a disulfide bridge upon the exposure to ROS and regulate the cytoplasm to nucleus transport. From sequence analysis and phylogenetic reconstruction of redox sensing states we propose that reactive cysteine acquisition through evolution leads to three separate E7s protein families that differ in the ROS sensing mechanism: non ROS-sensitive E7s; ROS-sensitive E7s using only a single or multiple reactive cysteine sensing mechanisms and ROS-sensitive E7s using a reactive-resolutive cysteine couple sensing mechanism.

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.

Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder.

Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage "Protein and nucleic acid structure and sequence analysis".

Ordered self-assembly mechanism of a spherical oncoprotein oligomer triggered by zinc removal and stabilized by an intrinsically disordered domain.

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.