Inflammasome regulation by adaptor isoforms, ASC and ASCb, via differential self-assembly.

ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular docking with the procaspase-1 CARD domain. Altogether, our results indicate that ASC's faster and less polydisperse polymerization is more efficient, plausibly explaining inflammasome activation differences by ASC isoforms at the molecular level.

Natural and engineered inflammasome adapter proteins reveal optimum linker length for self-assembly.

The inflammasome is a multiprotein complex that triggers the activation of proinflammatory cytokines. The adapter ASC and its isoform ASCb mediate inflammasome assembly via self-association and oligomerization with other inflammasome proteins by homotypic interactions of their two identical Death Domains, PYD and CARD, connected by a linker of different length: 23 (ASC) and 4 (ASCb) amino acids long. However, ASC is a more potent inflammasome activator compared to ASCb. Thus, adapter isoforms might be involved in the regulation of the inflammatory response. As previously reported, ASC's faster and less polydisperse self-association compared to ASCb points to interdomain flexibility resulting from the linker length as a key factor in inflammasome regulation. To test the influence of linker length in self-association, we have engineered the isoform ASC3X with identical PYD and CARD connected by a 69 amino acid-long linker (i.e., three-times longer than ASC's linker). Real-time NMR and dynamic light scattering data indicate that ASC3X polymerization is less effective and more polydisperse compared to ASC or ASCb. However, transmission electron micrographs show that ASC3X can polymerize into filaments. Comparative interdomain dynamics of the three isoforms obtained from NMR relaxation data reveal that ASCb tumbles as a rod, whereas the PYD and CARD of ASC and ASC3X tumble independently with marginally higher interdomain flexibility in ASC3X. Altogether, our data suggest that ASC's linker length is optimized for self-association by allowing enough flexibility to favor intermolecular homotypic interactions but simultaneously keeping both domains sufficiently close for essential participation in filament formation.

Underrepresented Impurities in 4-Hydroxyphenylpyruvate Affect the Catalytic Activity of Multiple Enzymes.

Macrophage migration inhibitory factor (MIF) is a key immunostimulatory protein with regulatory properties in several disorders, including inflammation and cancer. All the reported inhibitors that target the biological activities of MIF have been discovered by testing against its keto/enol tautomerase activity. While the natural substrate is still unknown, model MIF substrates are used for kinetic experiments. The most extensively used model substrate is 4-hydroxyphenyl pyruvate (4-HPP), a naturally occurring intermediate of tyrosine metabolism. Here, we examine the impact of 4-HPP impurities in the precise and reproducible determination of MIF kinetic data. To provide unbiased evaluation, we utilized 4-HPP powders from five different manufacturers. Biochemical and biophysical analyses showed that the enzymatic activity of MIF is highly influenced by underrepresented impurities found in 4-HPP. Besides providing inconsistent turnover results, the 4-HPP impurities also influence the accurate calculation of ISO-1's inhibition constant, an MIF inhibitor that is broadly used for in vitro and in vivo studies. The macromolecular NMR data show that 4-HPP samples from different manufacturers result in differential chemical shift perturbations of amino acids in MIF's active site. Our MIF-based conclusions were independently evaluated and confirmed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT); two additional enzymes that utilize 4-HPP as a substrate. Collectively, these results explain inconsistencies in previously reported inhibition values, highlight the effect of impurities on the accurate determination of kinetic parameters, and serve as a tool for designing error-free in vitro and in vivo experiments.

Natural and Engineered Isoforms of the Inflammasome Adaptor ASC Form Noncovalent, pH-Responsive Hydrogels.

The protein ASC polymerizes into intricate filament networks to assemble the inflammasome, a filamentous multiprotein complex that triggers the inflammatory response. ASC carries two Death Domains integrally involved in protein self-association for filament assembly. We have leveraged this behavior to create noncovalent, pH-responsive hydrogels of full-length, folded ASC by carefully controlling the pH as a critical factor in the polymerization process. We show that natural variants of ASC (ASC isoforms) involved in inflammasome regulation also undergo hydrogelation. To further demonstrate this general capability, we engineered proteins inspired by the ASC structure that also form hydrogels. We analyzed the structural network of the natural and engineered protein hydrogels using transmission and scanning electron microscopy and studied their viscoelastic behavior using shear rheology. Our results reveal one of the very few examples of hydrogels created by the self-assembly of globular proteins and domains in their native conformation and show that Death Domains can be used alone or as building blocks to engineer bioinspired hydrogels.

Structure, Activation and Regulation of NLRP3 and AIM2 Inflammasomes.

The inflammasome is a three-component (sensor, adaptor, and effector) filamentous signaling platform that shields from multiple pathogenic infections by stimulating the proteolytical maturation of proinflammatory cytokines and pyroptotic cell death. The signaling process initiates with the detection of endogenous and/or external danger signals by specific sensors, followed by the nucleation and polymerization from sensor to downstream adaptor and then to the effector, caspase-1. Aberrant activation of inflammasomes promotes autoinflammatory diseases, cancer, neurodegeneration, and cardiometabolic disorders. Therefore, an equitable level of regulation is required to maintain the equilibrium between inflammasome activation and inhibition. Recent advancement in the structural and mechanistic understanding of inflammasome assembly potentiates the emergence of novel therapeutics against inflammasome-regulated diseases. In this review, we have comprehensively discussed the recent and updated insights into the structure of inflammasome components, their activation, interaction, mechanism of regulation, and finally, the formation of densely packed filamentous inflammasome complex that exists as micron-sized punctum in the cells and mediates the immune responses.

The inflammasome adapter ASC assembles into filaments with integral participation of its two Death Domains, PYD and CARD.

The inflammasome is a multiprotein complex necessary for the onset of inflammation. The adapter protein ASC assembles inflammasome components by acting as a molecular glue between danger-signal sensors and procaspase-1. The assembly is mediated by ASC self-association and protein interactions via its two Death Domains, PYD and CARD. Truncated versions of ASC have been shown to form filaments, but information on the filaments formed by full-length ASC is needed to construct a meaningful model of inflammasome assembly. To gain insights into this system, we used a combination of transmission EM, NMR, and computational analysis to investigate intact ASC structures. We show that ASC forms ∼6-7-nm-wide filaments that stack laterally to form bundles. The structural characteristics and dimensions of the bundles indicate that both PYD and CARD are integral parts of the filament. A truncated version of ASC with only the CARD domain (ASCCARD) forms different filaments (∼3-4-nm width), providing further evidence that both domains work in concert in filament assembly. Ring-shaped protein particles bound to pre-existing filaments match the size of ASC dimer structures generated by NMR-based protein docking, suggesting that the ASC dimer could be a basic building block for filament formation. Solution NMR binding studies identified the protein surfaces involved in the ASCCARD-ASCCARD interaction. These data provide new insights into the structural underpinnings of the inflammasome and should inform future efforts to interrogate this important biological system.

Glutton: a tool for generating structural ensembles of partly disordered proteins from chemical shifts.

MOTIVATION: Many proteins are partially disordered in physiological conditions and only fold, fully or partially, upon binding. Their structural analysis is challenging because the accessible information, typically chemical shifts (CS) from nuclear magnetic resonance experiments, are averages over broad ensembles of conformations. We aim to develop a database for the analysis of such data in terms of conformational distributions of the protein backbone rather than of individual high-resolution structures. RESULTS: Glutton is the largest available database linking CS and protein 3D structures (5270 entries organized in three levels) and is searchable via a python script. It generates statistical distributions of ϕ-ψ dihedral angles based on CS or vice versa. Such ϕ-ψ distributions are used to calculate structural ensembles of partially disordered proteins from their CS. For folded proteins, such ensembles are excellent starting points for further refinement with additional experimental restraints (structure determination) or computational methods (structure prediction). AVAILABILITY AND IMPLEMENTATION: Glutton is freely available at https://github.com/YeeHo/Glutton. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structure, interactions and self-assembly of ASC-dependent inflammasomes.

The inflammasome is a multi-protein platform that assembles upon the presence of cues derived from infection or tissue damage, and triggers the inflammatory response. Inflammasome components include sensor proteins that detect danger signals, procaspase 1 and the adapter ASC (apoptosis-associated speck-like protein containing a CARD) tethering these molecules together. Upon inflammasome assembly, procaspase 1 self-activates and renders functional cytokines to arbitrate in the defense mechanism. This assembly is mediated by self-association and protein interactions via Death Domains. The inflammasome plays a critical role in innate immunity and its dysregulation is the culprit of many autoimmune disorders. An in-depth understanding of the factors involved in inflammasome assembly could help fight these conditions. This review describes our current knowledge on the biophysical aspects of inflammasome formation from the perspective of ASC. The specific characteristics of the three-dimensional solution structure and interdomain dynamics of ASC are explained in relation to its function in inflammasome assembly. Additionally, the review elaborates on the identification of ASC interacting surfaces at the amino acid level using NMR techniques. Finally, the macrostructures formed by full-length ASC and its two Death Domains studied with Transmission Electron Microscopy are compared in the context of a directional model for inflammasome assembly.

ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces.

Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASC·ASC and ASC·NLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling.

Interaction Networks in Protein Folding via Atomic-Resolution Experiments and Long-Time-Scale Molecular Dynamics Simulations.

The integration of atomic-resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone. Here, we combine equilibrium NMR measurements of thermal unfolding and long molecular dynamics simulations to investigate the folding of gpW, a protein with two-state-like, fast folding dynamics and cooperative equilibrium unfolding behavior. Experiments and simulations expose a remarkably complex pattern of structural changes that occur at the atomic level and from which the detailed network of residue-residue couplings associated with cooperative folding emerges. Such thermodynamic residue-residue couplings appear to be linked to the order of mechanistically significant events that take place during the folding process. Our results on gpW indicate that the methods employed in this study are likely to prove broadly applicable to the fine analysis of folding mechanisms in fast folding proteins.

Assembly mechanism of the inflammasome sensor AIM2 revealed by single molecule analysis.

Pathogenic dsDNA prompts AIM2 assembly leading to the formation of the inflammasome, a multimeric complex that triggers the inflammatory response. The recognition of foreign dsDNA involves AIM2 self-assembly concomitant with dsDNA binding. However, we lack mechanistic and kinetic information on the formation and propagation of the assembly, which can shed light on innate immunity's time response and specificity. Combining optical traps and confocal fluorescence microscopy, we determine here the association and dissociation rates of the AIM2-DNA complex at the single molecule level. We identify distinct mechanisms for oligomer growth via the binding of incoming AIM2 molecules to adjacent dsDNA or direct interaction with bound AIM2 assemblies, resembling primary and secondary nucleation. Through these mechanisms, the size of AIM2 oligomers can increase fourfold in seconds. Finally, our data indicate that single AIM2 molecules do not diffuse/scan along the DNA, suggesting that oligomerization depends on stochastic encounters with DNA and/or DNA-bound AIM2.

Natural and engineered isoforms of the inflammasome adaptor ASC form non-covalent, pH-responsive hydrogels.

The protein ASC polymerizes into intricate filament networks to assemble the inflammasome, a filamentous multiprotein complex that triggers the inflammatory response. ASC carries two Death Domains integrally involved in protein self-association for filament assembly. We have leveraged this behavior to create non-covalent, pH-responsive hydrogels of full-length, folded ASC by carefully controlling the pH as a critical factor in the polymerization process. We show that natural variants of ASC (ASC isoforms) involved in inflammasome regulation also undergo hydrogelation. To further demonstrate this general capability, we engineered proteins inspired in the ASC structure that successfully form hydrogels. We analyzed the structural network of the natural and engineered protein hydrogels using transmission and scanning electron microscopy, and studied their viscoelastic behavior by shear rheology. Our results reveal one of the very few examples of hydrogels created by the self-assembly of globular proteins and domains in their native conformation and show that Death Domains can be used alone or as building blocks to engineer bioinspired hydrogels.

Nuclear magnetic resonance study of protein-protein interactions involving apoptosis regulator Diva (Boo) and the BH3 domain of proapoptotic Bcl-2 members.

According to biochemical assays, the Bcl-2 protein Diva from mouse regulates programmed cell death by heterodimerizing with other members of the family and by interacting with the apoptotic protease-activating factor Apaf-1. In typical Bcl-2 heterodimers, peptide fragments comprising the Bcl-2 homology domain 3 (BH3 domain) of proapoptotic members are capable of forming functional complexes with prosurvival proteins. High-resolution structural studies have revealed that the BH3 peptide forms an α-helix positioned in a canonical hydrophobic cleft of the antiapoptotic protein. Because Diva shows mutations in conserved residues within this area, it has been proposed to have a different interacting surface. However, we showed previously that Diva binds through the canonical groove the BH3 peptide of the human Bcl-2 killing member Harakiri. To further test Diva's binding capabilities, here we show Nuclear Magnetic Resonance (NMR) data, indicating that Diva binds peptides derived from the BH3 domain of several other proapoptotic Bcl-2 proteins, including mouse Harakiri, Bid, Bak and Bmf. We have measured the binding affinities of the heterodimers, which show significant variability. Structural models of the protein-peptide complexes based on NMR chemical shift perturbation data indicate that the binding surface is analogous. These models do not rely on NMR NOE (Nuclear Overhauser Effect) data, and thus our results can only suggest that the complexes share similar intermolecular interactions. However, the observed affinity differences correlate with the α-helical population of the BH3-peptides obtained from circular dichroism experiments, which highlights a role of conformational selection in the binding mechanism. Altogether, our results shed light on important factors governing Diva-BH3 peptide molecular recognition mode.

A designed protein as experimental model of primordial folding.

How do proteins accomplish folding during early evolution? Theoretically the mechanism involves the selective stabilization of the native structure against all other competing compact conformations in a process that involves cumulative changes in the amino acid sequence along geological timescales. Thus, an evolved protein folds into a single structure at physiological temperature, but the conformational competition remains latent. For natural proteins such competition should emerge only near cryogenic temperatures, which places it beyond experimental testing. Here, we introduce a designed monomeric miniprotein (FSD-1ss) that within biological temperatures (330-280 K) switches between simple fast folding and highly complex conformational dynamics in a structurally degenerate compact ensemble. Our findings demonstrate the physical basis for protein folding evolution in a designed protein, which exhibits poorly evolved or primordial folding. Furthermore, these results open the door to the experimental exploration of primitive folding and the switching between alternative protein structures that takes place in evolutionary branching points and prion diseases, as well as the benchmarking of de novo design methods.

NMR Analysis of Protein Folding Interaction Networks.

Theory and experimental evidence unequivocally indicate that protein folding is far more complex than the two-state (all-or-none) model that is usually assumed in the analysis of folding experiments. Proteins tend to fold hierarchically by forming secondary structure elements, followed by supersecondary arrangements, and other intermediate states that ultimately adopt the native tertiary fold as a result of a delicate balance between interatomic interactions and entropic contributions. However, small proteins with simple folds typically follow downhill folding, characterized by very small energetic barriers (<3 RT) that allow multiple protein conformations to be populated along the folding path down the free energy landscape, reaching the native fold at the lowest energy level.Here we describe the use of solution-state nuclear magnetic resonance (NMR) for the analysis of protein folding interaction networks at atomic resolution. The assignment of NMR spectra acquired at different unfolding conditions provides hundreds of atomic unfolding curves that are analyzed to infer the network of folding interactions. The method is particularly useful to study small proteins that fold autonomously in the sub-millisecond timescale. The information obtained from the application of this method can potentially unveil the basic relationships between protein structure and folding.

Quantitative Studies on the Interaction between Saposin-like Proteins and Synthetic Lipid Membranes.

Members of the saposin-fold protein family and related proteins sharing a similar fold (saposin-like proteins; SAPLIP) are peripheral-membrane binding proteins that perform essential cellular functions. Saposins and SAPLIPs are abundant in both plant and animal kingdoms, and peripherally bind to lipid membranes to play important roles in lipid transfer and hydrolysis, defense mechanisms, surfactant stabilization, and cell proliferation. However, quantitative studies on the interaction between proteins and membranes are challenging due to the different nature of the two components in relation to size, structure, chemical composition, and polarity. Using liposomes and the saposin-fold member saposin C (sapC) as model systems, we describe here a method to apply solution NMR and dynamic light scattering to study the interaction between SAPLIPs and synthetic membranes at the quantitative level. Specifically, we prove with NMR that sapC binds reversibly to the synthetic membrane in a pH-controlled manner and show the dynamic nature of its fusogenic properties with dynamic light scattering. The method can be used to infer the optimal pH for membrane binding and to determine an apparent dissociation constant (KDapp) for protein-liposome interaction. We propose that these experiments can be applied to other proteins sharing the saposin fold.

Engineering of Saposin C Protein Chimeras for Enhanced Cytotoxicity and Optimized Liposome Binding Capability.

Saposin C (sapC) is a lysosomal, peripheral-membrane protein displaying liposome fusogenic capabilities. Proteoliposomes of sapC and phosphatidylserine have been shown to be toxic for cancer cells and are currently on clinical trial to treat glioblastoma. As proof-of-concept, we show two strategies to enhance the applications of sapC proteoliposomes: (1) Engineering chimeras composed of sapC to modulate proteoliposome function; (2) Engineering sapC to modify its lipid binding capabilities. In the chimera design, sapC is linked to a cell death-inducing peptide: the BH3 domain of the Bcl-2 protein PUMA. We show by solution NMR and dynamic light scattering that the chimera is functional at the molecular level by fusing liposomes and by interacting with prosurvival Bcl-xL, which is PUMA's known mechanism to induce cell death. Furthermore, sapC-PUMA proteoliposomes enhance cytotoxicity in glioblastoma cells compared to sapC. Finally, the sapC domain of the chimera has been engineered to optimize liposome binding at pH close to physiological values as protein-lipid interactions are favored at acidic pH in the native protein. Altogether, our results indicate that the properties of sapC proteoliposomes can be modified by engineering the protein surface and by the addition of small peptides as fusion constructs.

Structure and interdomain dynamics of apoptosis-associated speck-like protein containing a CARD (ASC).

The human protein ASC is a key mediator in apoptosis and inflammation. Through its two death domains (pyrin and CARD) ASC interacts with cell death executioners, acts as an essential adapter for inflammasome integrity, and oligomerizes into functional supramolecular assemblies. However, these functions are not understood at the structural-dynamic level. This study reports the solution structure and interdomain dynamics of full-length ASC. The pyrin and CARD domains are structurally independent six-helix bundle motifs connected by a 23-residue linker. The CARD structure reveals two distinctive characteristics; helix 1 is not fragmented as in all other known CARDs, and its electrostatic surface shows a uniform distribution of positive and negative charges, whereas these are commonly separated into two areas in other death domains. The linker adopts residual structure resulting in a back-to-back orientation of the domains, which avoids steric interference of each domain with the binding site of the other. NMR relaxation experiments show that the linker is flexible despite the residual structure. This flexibility could help expand the relative volume occupied by each domain, thus increasing the capture radius for effectors. Based on the ASC structure, a tentative model is proposed to illustrate how ASC oligomerizes via CARD and pyrin homophilic interactions. Moreover, ASC oligomers have been analyzed by atomic force microscopy, showing a predominant species of disk-like particles of approximately 12-nm diameter and approximately 1-nm height. Taken together, these results provide structural insight into the behavior of ASC as an adapter molecule.

Conformational Priming of RepA-WH1 for Functional Amyloid Conversion Detected by NMR Spectroscopy.

How proteins with a stable globular fold acquire the amyloid state is still largely unknown. RepA, a versatile plasmidic DNA binding protein from Pseudomonas savastanoi, is functional as a transcriptional repressor or as an initiator or inhibitor of DNA replication, the latter via assembly of an amyloidogenic oligomer. Its N-terminal domain (WH1) is responsible for discrimination between these functional abilities by undergoing insufficiently understood structural changes. RepA-WH1 is a stable dimer whose conformational dynamics had not been explored. Here, we have studied it through NMR {1H}-15N relaxation and H/D exchange kinetics measurements. The N- and the C-terminal α-helices, and the internal amyloidogenic loop, are partially unfolded in solution. S4-indigo, a small inhibitor of RepA-WH1 amyloidogenesis, binds to and tethers the N-terminal α-helix to a ß-hairpin that is involved in dimerization, thus providing evidence for a priming role of fraying ends and dimerization switches in the amyloidogenesis of folded proteins.

Protein interactions of the inflammasome adapter ASC by solution NMR.

ASC (apoptosis-associated speck-like protein containing a CARD) is a modular protein that functions as an adapter of the inflammasome, a multi-protein complex that triggers the inflammatory response in the presence of infection or cell damage. ASC bridges the inflammasome components (PYD-containing sensors and procaspase 1) via homotypic interactions mediated by its two death domains, PYD and CARD. The self-assembly and oligomerization of multiple copies of these three proteins result in the activation of procaspase 1, in turn rendering different cytokines functional. An in-depth understanding of ASC binding capabilities is crucial to decipher the molecular mechanisms of its role in inflammasome formation. In this chapter, we discuss the use of solution NMR to identify specific interacting surfaces of the inflammasome adapter ASC, and describe detailed protocols to perform NMR titrations with Death Domains to obtain apparent dissociation constants of the resulting complexes. The incorporation of NMR restraints in molecular docking to obtain models of these protein assemblies is presented.