Background: RAGE is a multiligand cell surface receptor involved in various human diseases. Results: The solution structure of human sRAGE was determined. sRAGE oligomerization is concentration-and calcium-dependent. Conclusion:The monomer displays a J-like shape; the dimer is elongated and forms through association of two N-terminal domains. Significance: This paves the way for the design of new therapeutic strategies for a large number of pathologies.

Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.

Picornaviruses are small pathogen RNA viruses, like poliovirus, hepatitis A virus, rhinovirus, and others. They produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornains 2A and 3C. Picornain 3C represents an intermediate between the serine peptidase chymotrypsin and the cysteine peptidase papain. Its steric structure resembles chymotrypsin, but its nucleophile is a thiol instead of the hydroxyl group. The histidine is a general base catalyst in chymotrypsin but forms a thiolate-imidazolium ion pair in papain. The third member of the catalytic triad is an acid (Glu71) as in chymotrypsin rather than an amide found in papain. Transformation of poliovirus 3C peptidase into a serine peptidase results in lower activity by a factor of 430, but the activity extends toward higher pH with the more basic hydroxyl group. The decrease in activity is caused by the less ordered active site, as supported by the unfavorable entropy of activation. At 25 degrees C the specificity rate constant for the thiol enzyme approaches k(1), the rate constant for the formation of the enzyme-substrate complex, but k(2), the acylation constant, becomes predominant with the increase in temperature. In contrast, for the serine peptidase the specificity constant is less than k(1) over the entire temperature range, and the transition state is controlled by both k(1) and k(2). The acidic component of the catalytic triad is essential for activity, but its negative charge does not influence the ionization of the thiol group.

BackgroundArabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL.ResultsThe TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein.ConclusionsThe Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

Drug discovery frequently relies on the kinetic analysis of physicochemical reactions that are at the origin of the disease state. Amyloid fibril formation has been extensively investigated in relation to prevalent and rare neurodegenerative diseases, but thus far no therapeutic solution has directly arisen from this knowledge. Other aggregation pathways producing smaller, hard-to-detect soluble oligomers are increasingly appointed as the main reason for cell toxicity and cell-to-cell transmissibility. Here we show that amyloid fibrillation kinetics can be used to unveil the protein oligomerization state. This is illustrated for human insulin and ataxin-3, two model proteins for which the amyloidogenic and oligomeric pathways are well characterized. Aggregation curves measured by the standard thioflavin-T (ThT) fluorescence assay are shown to reflect the relative composition of protein monomers and soluble oligomers measured by nuclear magnetic resonance (NMR) for human insulin, and by dynamic light scattering (DLS) for ataxin-3. Unconventional scaling laws of kinetic measurables were explained using a single set of model parameters consisting of two rate constants, and in the case of ataxin-3, an additional order-of-reaction. The same fitted parameters were used in a discretized population balance that adequately describes time-course measurements of fibril size distributions. Our results provide the opportunity to study oligomeric targets using simple, high-throughput compatible, biophysical assays.

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g., for the design of better drug screening strategies, or be compiled in databases for benchmarking novel aggregation-predicting algorithms. Given that minute protocol variations determine different outcomes of protein aggregation assays, there is a strong urge for standardized descriptions of the different types of aggregates and the detailed methods used in their production. In an attempt to address this need, we assembled the Minimum Information Required for Reproducible Aggregation Experiments (MIRRAGGE) guidelines, considering first-principles and the established literature on protein self-assembly and aggregation. This consensus information aims to cover the major and subtle determinants of experimental reproducibility while avoiding excessive technical details that are of limited practical interest for non-specialized users. The MIRRAGGE table (template available in Supplementary Information) is useful as a guide for the design of new studies and as a checklist during submission of experimental reports for publication. Full disclosure of relevant information also enables other researchers to reproduce results correctly and facilitates systematic data deposition into curated databases.

This work combined water equilibration fundamentals of vapor diffusion crystallization techniques with protein solubility data in order to obtain the variation of protein supersaturation throughout the protein crystallization assays. Once the supersaturation build up profiles (SBUPs) are known, the wide screening space of crystallization conditions is reduced to the key variable for crystal formation and growth, which is supersaturation and its variation with time. Our previous water equilibration model was expanded to include the case of drop evaporation at constant contact area during the hanging drop method. Crystallization experiments of lysozyme were performed under different experimental conditions and the results were interpreted according to the respective SBPUs. In particular, the number and size of the crystals were evaluated at the moment of the SBUP that corresponded to their formation. Following this methodology, two nucleation behaviors were identified depending on the supersaturation levels at which crystal formation occurs. These behaviors, which are believed to be closely linked with the diffracting properties of the crystals, are dictated not only by classic thermodynamic and kinetic factors affecting crystallization and water equilibration, but also by phenomena related to the drop preparation procedures.

Picornains 2A are cysteine proteases of picornaviruses, a virus family containing several human and animal pathogens. The pH dependencies of the alkylations of picornain 2A of rhinovirus type 2 with iodoacetamide and iodoacetate show two reactive thiol forms, namely the free thiolate ion at high pH and an imidazole assisted thiol group at low pH. Kinetic deuterium isotope effects do not support general base catalysis by the imidazole group, but rather the existence of a catalytically competent thiolate^imidazolium ion-pair. The nature of the ionpair differs from that of papain, the paradigm of cysteine proteases. The ion-pair is confined to the same, unusually narrow pH range in which the enzyme exhibits catalytic activity. ß