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.

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.

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.

On the Differential Roles of Mg2+, Zn2+, and Cu2+ in the Equilibrium of ß-N-Methyl-Amino-L-Alanine (BMAA) and its Carbamates.

ß-N-methyl-amino-L-alanine (BMAA) in the presence of bicarbonate (HCO3-) undergoes structural modifications generating two carbamate species, α-carbamate and ß-carbamate forms of BMAA. The chemical structure of BMAA and BMAA-carbamate adducts strongly suggest they may interact with divalent metal ions. The ability of BMAA to cross the blood-brain barrier and possibly interact with divalent metal ions may augment the neurotoxicity of these molecules. To understand the effects of divalent metal ions (Mg2+, Zn2+, and Cu2+) on the overall dynamic equilibrium between BMAA and its carbamate adducts, a systematic study using nuclear magnetic resonance (NMR) is presented. The chemical equilibria between BMAA, its carbamate adducts, and each of the divalent ions were studied using two-dimensional chemical exchange spectroscopy (EXSY). The NMR results demonstrate that BMAA preferentially interacts with Zn2+ and Cu2+, causing an overall reduction in the production of carbamate species by altering the dynamic equilibria. The NMR-based spectral changes due to the BMAA interaction with Cu2+ is more drastic than with the Zn2+, under the same stoichiometric ratios of BMAA and the individual divalent ions. However, the presence of Mg2+ does not significantly alter the dynamic equilibria between BMAA and its carbamate adducts. The NMR-based results are further validated using circular dichroism (CD) spectroscopy, observing the n ➔ π interaction in the complex formation of BMAA and the divalent metal ions, with additional verification of the interaction with Cu2+ using UV-Vis spectroscopy. Our results demonstrate that BMAA differentially interacts with divalent metal ions (Mg2+ < Zn2+ < Cu2+), and thus alters the rate of formation of carbamate products. The equilibria between BMAA, the bicarbonate ions, and the divalent metal ions may alter the total population of a specific form of BMAA-ion complex at physiological conditions and, therefore, add a level of complexity of the mechanisms by which BMAA acts as a neurotoxin.

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.

Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts.

Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms.