NAGPKin: Nucleation-and-growth parameters from the kinetics of protein phase separation.

The assembly of biomolecular condensate in eukaryotic cells and the accumulation of amyloid deposits in neurons are processes involving the nucleation and growth (NAG) of new protein phases. To therapeutically target protein phase separation, drug candidates are tested in in vitro assays that monitor the increase in the mass or size of the new phase. Limited mechanistic insight is, however, provided if empirical or untestable kinetic models are fitted to these progress curves. Here we present the web server NAGPKin that quantifies NAG rates using mass-based or size-based progress curves as the input data. A report is generated containing the fitted NAG parameters and elucidating the phase separation mechanisms at play. The NAG parameters can be used to predict particle size distributions of, for example, protein droplets formed by liquid-liquid phase separation (LLPS) or amyloid fibrils formed by protein aggregation. Because minimal intervention is required from the user, NAGPKin is a good platform for standardized reporting of LLPS and protein self-assembly data. NAGPKin is useful for drug discovery as well as for fundamental studies on protein phase separation. NAGPKin is freely available (no login required) at https://nagpkin.i3s.up.pt.

Pathogen-specific structural features of Candida albicans Ras1 activation complex: uncovering new antifungal drug targets.

An important feature associated with Candida albicans pathogenicity is its ability to switch between yeast and hyphal forms, a process in which CaRas1 plays a key role. CaRas1 is activated by the guanine nucleotide exchange factor (GEF) CaCdc25, triggering hyphal growth-related signaling pathways through its conserved GTP-binding (G)-domain. An important function in hyphal growth has also been proposed for the long hypervariable region downstream the G-domain, whose unusual content of polyglutamine stretches and Q/N repeats make CaRas1 unique within Ras proteins. Despite its biological importance, both the structure of CaRas1 and the molecular basis of its activation by CaCdc25 remain unexplored. Here, we show that CaRas1 has an elongated shape and limited conformational flexibility and that its hypervariable region contains helical structural elements, likely forming an intramolecular coiled-coil. Functional assays disclosed that CaRas1-activation by CaCdc25 is highly efficient, with activities up to 2,000-fold higher than reported for human GEFs. The crystal structure of the CaCdc25 catalytic region revealed an active conformation for the α-helical hairpin, critical for CaRas1-activation, unveiling a specific region exclusive to CTG-clade species. Structural studies on CaRas1/CaCdc25 complexes also revealed an interaction surface clearly distinct from that of homologous human complexes. Furthermore, we identified an inhibitory synthetic peptide, prompting the proposal of a key regulatory mechanism for CaCdc25. To our knowledge, this is the first report of specific inhibition of the CaRas1-activation via targeting its GEF. This, together with their unique pathogen-structural features, disclose a set of novel strategies to specifically block this important virulence-related mechanism. IMPORTANCE Candida albicans is the main causative agent of candidiasis, the commonest fungal infection in humans. The eukaryotic nature of C. albicans and the rapid emergence of antifungal resistance raise the challenge of identifying novel drug targets to battle this prevalent and life-threatening disease. CaRas1 and CaCdc25 are key players in the activation of signaling pathways triggering multiple virulence traits, including the yeast-to-hypha interconversion. The structural similarity of the conserved G-domain of CaRas1 to those of human homologs and the lack of structural information on CaCdc25 has impeded progress in targeting these proteins. The unique structural and functional features for CaRas1 and CaCdc25 presented here, together with the identification of a synthetic peptide capable of specifically inhibiting the GEF activity of CaCdc25, open new possibilities to uncover new antifungal drug targets against C. albicans virulence.

Drug repurposing of dopaminergic drugs to inhibit ataxin-3 aggregation.

The accumulation of mutant ataxin-3 (Atx3) in neuronal nuclear inclusions is a pathological hallmark of Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia Type 3. Decreasing the protein aggregation burden is a possible disease-modifying strategy to tackle MJD and other neurodegenerative disorders for which only symptomatic treatments are currently available. We performed a drug repurposing screening to identify inhibitors of Atx3 aggregation with known toxicological and pharmacokinetic profiles. Interestingly, dopamine hydrochloride and other catecholamines are among the most potent inhibitors of Atx3 aggregation in vitro. Our results indicate that low micromolar concentrations of dopamine markedly delay the formation of mature amyloid fibrils of mutant Atx3 through the inhibition of the earlier oligomerization steps. Although dopamine itself does not cross the blood-brain barrier, dopamine levels in the brain can be increased by low doses of dopamine precursors and dopamine agonists commonly used to treat Parkinsonian symptoms. In agreement, treatment with levodopa ameliorated motor symptoms in a C. elegans model of MJD. These findings suggest a possible application of dopaminergic drugs to halt or reduce Atx3 accumulation in the brains of MJD patients.

Quantification of Surface Tension Effects and Nucleation-and-Growth Rates during Self-Assembly of Biological Condensates.

Liquid-solid and liquid-liquid phase separation (PS) drives the formation of functional and disease-associated biological assemblies. Principles of phase equilibrium are here employed to derive a general kinetic solution that predicts the evolution of the mass and size of biological assemblies. Thermodynamically, protein PS is determined by two measurable concentration limits: the saturation concentration and the critical solubility. Due to surface tension effects, the critical solubility can be higher than the saturation concentration for small, curved nuclei. Kinetically, PS is characterized by the primary nucleation rate constant and a combined rate constant accounting for growth and secondary nucleation. It is demonstrated that the formation of a limited number of large condensates is possible without active mechanisms of size control and in the absence of coalescence phenomena. The exact analytical solution can be used to interrogate how the elementary steps of PS are affected by candidate drugs.

MIRRAGGE - Minimum Information Required for Reproducible AGGregation Experiments.

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.

Genetic code ambiguity modulates the activity of a C. albicans MAP kinase linked to cell wall remodeling.

The human fungal pathogen Candida albicans ambiguously decodes the universal leucine CUG codon predominantly as serine but also as leucine. C. albicans has a high capacity to survive and proliferate in adverse environments but the rate of leucine incorporation fluctuates in response to different stress conditions. C. albicans is adapted to tolerate this ambiguous translation through a mechanism that combines drastic decrease in CUG usage and reduction of CUG-encoded residues in conserved positions in the protein sequences. However, in a few proteins, the residues encoded by CUG codons are found in strictly conserved positions, suggesting that this genetic code alteration might have a functional impact. One such example is Cek1, a central signaling protein kinase that contains a single CUG-encoded residue at a conserved position, whose identity might regulate the correct flow of information across the MAPK cascade. Here we show that insertion of a leucine at the CUG-encoded position decreases the stability of Cek1, apparently without major structural alterations. In contrast, incorporation of a serine residue at the CUG position induces the autophosphorylation of the conserved tyrosine residue of the Cek1 231TEY233 motif, and increases its intrinsic kinase activity in vitro. These findings show that CUG ambiguity modulates the activity of Cek1, a key kinase directly linked to morphogenesis and virulence in C. albicans.

Chemical Kinetic Strategies for High-Throughput Screening of Protein Aggregation Modulators.

Insoluble aggregates staining positive to amyloid dyes are known histological hallmarks of different neurodegenerative disorders and of type II diabetes. Soluble oligomers are smaller assemblies whose formation prior to or concomitant with amyloid deposition has been associated to the processes of disease propagation and cell death. While the pathogenic mechanisms are complex and differ from disease to disease, both types of aggregates are important biological targets subject to intense investigation in academia and industry. Here we review recent advances in the fundamental understanding of protein aggregation that can be used on the development of anti-amyloid and anti-oligomerization drugs. Specifically, we pinpoint the chemical kinetic aspects that should be attended during the development of high-throughput screening assays and in the hit validation phase. The strategies here devised are expected to establish a connection between basic research and pharmaceutical innovation.

Anti-TTR Nanobodies Allow the Identification of TTR Neuritogenic Epitope Associated with TTR-Megalin Neurotrophic Activities.

Transthyretin (TTR) has intrinsic neurotrophic physiological activities independent from its thyroxine ligands, which involve activation of signaling pathways through interaction with megalin. Still, the megalin binding motif on TTR is unknown. Nanobodies (Nb) have the ability to bind "hard to reach" epitopes being useful tools for protein/structure function. In this work, we characterize two anti-TTR Nanobodies, with similar mouse TTR binding affinities, although only one is able to block its neuritogenic activity (169F7_Nb). Through epitope mapping, we identified amino acids 14-18, at the entrance of the TTR central channel, to be important for interaction with megalin, and a stable TTR K15N mutant in that region was constructed. The TTR K15N mutant lacks neuritogenic activity, indicating that K15 is critical for TTR neuritogenic activity. Thus, we identify the putative binding site for megalin and describe two Nanobodies that will allow research and clarification of TTR physiological properties, regarding its neurotrophic effects.

Probing the Occurrence of Soluble Oligomers through Amyloid Aggregation Scaling Laws.

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.

Ser or Leu: structural snapshots of mistranslation in Candida albicans.

Candida albicans is a polymorphic opportunistic fungal pathogen normally residing as commensal on mucosal surfaces, skin and gastrointestinal and genitourinary tracts. However, in immunocompromised patients C. albicans can cause superficial mucosal infections or life-threatening disseminated candidemia. A change in physiological conditions triggers a cascade of molecular events leading to morphogenetic alterations and increased resistance to damage induced by host defenses. The complex biology of this human pathogen is reflected in its morphological plasticity and reinforced by the ability to ambiguously translate the universal leucine CUG codon predominantly as serine, but also as leucine. Mistranslation affects more than half of C. albicans proteome and it is widespread across many biological processes. A previous analysis of CTG-codon containing gene products in C. albicans suggested that codon ambiguity subtly shapes protein function and might have a pivotal role in signaling cascades associated with morphological changes and pathogenesis. In this review we further explore this hypothesis by highlighting the role of ambiguous decoding in macromolecular recognition of key effector proteins associated with the regulation of signal transduction cascades and the cell cycle, which are critical processes for C. albicans morphogenic plasticity under a variety of environmental conditions.

Solution structure of the soluble receptor for advanced glycation end products (sRAGE).

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor involved in various human diseases, as it binds to numerous molecules and proteins that modulate the activity of other proteins. Elucidating the three-dimensional structure of this receptor is therefore most important for understanding its function during activation and cellular signaling. The major alternative splice product of RAGE comprises its extracellular region that occurs as a soluble protein (sRAGE). Although the structures of sRAGE domains were available, their assembly into the functional full-length protein remained unknown. We observed that the protein has concentration-dependent oligomerization behavior, and this is also mediated by the presence of Ca(2+) ions. Moreover, using synchrotron small angle x-ray scattering, the solution structure of human sRAGE was determined in the monomeric and dimeric forms. The model for the monomer displays a J-like shape, whereas the dimer is formed through the association of the two N-terminal domains and has an elongated structure. These results provide insights into the assembly of the RAGE homodimer, which is essential for signal transduction, and the sRAGE:RAGE heterodimer that leads to blockage of the receptor signaling, paving the way for the design of therapeutic strategies for a large number of different pathologies.

Functional characterization of Arabidopsis thaliana transthyretin-like protein.

BACKGROUND: Arabidopsis 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. RESULTS: The 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. CONCLUSIONS: The 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.

The unusual catalytic triad of poliovirus protease 3C.

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.