Partitioning conformational intermediates between competing refolding and aggregation pathways: insights into transthyretin amyloid disease.
ABSTRACT: Amyloid diseases are caused by the aberrant assembly of a protein in the extracellular space. Folded proteins are not amyloidogenic; however, the native state is generally in equilibrium with a minor population of unfolded or partially folded aggregation-competent conformers outside of the cell. Understanding how the partially unfolded conformers kinetically partition between the competing refolding and aggregation pathways provides insight into how misfolding, which occurs continuously, becomes pathogenic. Towards this end, we have previously studied the amyloidogenicity of transthyretin (TTR), a human beta-sheet-rich homotetrameric protein that must undergo rate-limiting tetramer dissociation and partial monomer unfolding to misassemble into amyloid and other aggregates. We demonstrate herein that TTR homotetramers reassemble by an unusual monomer-dimer-trimer-tetramer (MDRT) pathway. Therefore, the rate of every step in the reassembly pathway is dependent on the concentration of folded TTR monomer. Partitioning soluble TTR monomers between the reassembly pathway and the aggregation pathway should therefore depend on the relative concentrations of aggregates and assembly intermediates. Aggregate clearance is envisioned to play an important role in the partitioning of protein in vivo, where partitioning to the aggregation pathway becomes increasingly favorable under conditions where the concentration of aggregates is increased because aggregate clearance is slow relative to the rate of aggregation. This shift from efficient to inefficient aggregate clearance could occur with aging, offering an explanation for the age-associated nature of these neurodegenerative diseases.
Project description:Glycosaminoglycans (GAGs), which are found in association with all extracellular amyloid deposits in humans, are known to accelerate the aggregation of various amyloidogenic proteins in vitro. However, the precise molecular mechanism(s) by which GAGs accelerate amyloidogenesis remains elusive. Herein, we show that sulfated GAGs, especially heparin, accelerate transthyretin (TTR) amyloidogenesis by quaternary structural conversion. The clustering of sulfate groups on heparin and its polymeric nature are essential features for accelerating TTR amyloidogenesis. Heparin does not influence TTR tetramer stability or TTR dissociation kinetics, nor does it alter the folded monomer-misfolded monomer equilibrium directly. Instead, heparin accelerates the conversion of preformed TTR oligomers into larger aggregates. The more rapid disappearance of monomeric TTR in the presence of heparin likely reflects the fact that the monomer-misfolded amyloidogenic monomer-oligomer-TTR fibril equilibria are all linked, a hypothesis that is strongly supported by the light scattering data. TTR aggregates prepared in the presence of heparin exhibit a higher resistance to trypsin and proteinase K proteolysis and a lower exposure of hydrophobic side chains comprising hydrophobic clusters, suggesting an active role for heparin in amyloidogenesis. Our data suggest that heparin accelerates TTR aggregation by a scaffold-based mechanism, in which the sulfate groups comprising GAGs interact primarily with TTR oligomers through electrostatic interactions, concentrating and orienting the oligomers, facilitating the formation of higher molecular weight aggregates. This model raises the possibility that GAGs may play a protective role in human amyloid diseases by interacting with proteotoxic oligomers and promoting their association into less toxic amyloid fibrils.
Project description:Aggregation of Cu, Zn superoxide dismutase (SOD1) is often found in amyotrophic lateral sclerosis patients. The fibrillar aggregates formed by wild type and various disease-associated mutants have recently been found to have distinct cores and morphologies. Previous computational and experimental studies of wild-type SOD1 suggest that the apo-monomer, highly aggregation prone, displays substantial local unfolding dynamics. The residual folded structure of locally unfolded apoSOD1 corresponds to peptide segments forming the aggregation core as identified by a combination of proteolysis and mass spectroscopy. Therefore, we hypothesize that the destabilization of apoSOD1 caused by various mutations leads to distinct local unfolding dynamics. The partially unfolded structure, exposing the hydrophobic core and backbone hydrogen bond donors and acceptors, is prone to aggregate. The peptide segments in the residual folded structures form the "building block" for aggregation, which in turn determines the morphology of the aggregates. To test this hypothesis, we apply a multiscale simulation approach to study the aggregation of three typical SOD1 variants: wild type, G37R, and I149T. Each of these SOD1 variants has distinct peptide segments forming the core structure and features different aggregate morphologies. We perform atomistic molecular dynamics simulations to study the conformational dynamics of apoSOD1 monomer and coarse-grained molecular dynamics simulations to study the aggregation of partially unfolded SOD1 monomers. Our computational studies of monomer local unfolding and the aggregation of different SOD1 variants are consistent with experiments, supporting the hypothesis of the formation of aggregation "building blocks" via apo-monomer local unfolding as the mechanism of SOD1 fibrillar aggregation.
Project description:Transthyretin (TTR) is a tetrameric serum protein associated with multiple systemic amyloid diseases. In these disorders, TTR aggregates in extracellular environments through a mechanism involving rate-limiting dissociation of the tetramer to monomers, which then misfold and aggregate into soluble oligomers and amyloid fibrils that induce toxicity in distal tissues. Using an assay established herein, we show that highly destabilized, aggregation-prone TTR variants are secreted as both native tetramers and non-native conformations that accumulate as high-molecular-weight oligomers. Pharmacologic chaperones that promote endoplasmic reticulum (ER) proteostasis of destabilized TTR variants increase their fraction secreted as a tetramer and reduce extracellular aggregate populations. In contrast, disrupting ER proteostasis reduces the fraction of destabilized TTR secreted as a tetramer and increases extracellular aggregates. These results identify ER proteostasis as a factor that can affect conformational integrity and thus toxic aggregation of secreted amyloidogenic proteins associated with the pathology of protein aggregation diseases.
Project description:Aggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration, aggregation-prone intermediates are challenging to probe using conventional assays. We report a 19F-NMR assay that leverages a highly sensitive trifluoroacetyl probe at a strategic site that gives distinct 19F chemical shifts for the TTR tetramer and monomeric intermediate and enables direct quantification of their populations during the aggregation process. Integration of real-time 19F-NMR and turbidity measurements as a function of temperature allows kinetic and mechanistic dissection of the aggregation pathway of both wild-type and mutant TTR. At physiological temperature, the monomeric intermediate formed by wild-type TTR under mildly acidic conditions rapidly aggregates into species that are invisible to NMR, leading to loss of the NMR signal at the same rate as the turbidity increase. Lower temperature accelerates tetramer dissociation and decelerates monomer tetramerization and oligomerization via reduced hydrophobic interactions associated with packing of a phenylalanine (F87) into a neighboring protomer. As a result, the intermediate accumulates to a higher level, and formation of higher-order aggregates is delayed. Application of this assay to pathogenic (V30M, L55P, and V122I) and protective (T119M) mutants revealed significant differences in behavior. A monomeric intermediate was observed only for V122I: aggregation of V30M and L55P proceeds without an observable monomeric intermediate, whereas the protective mutant T119M remains resistant to tetramer dissociation and aggregation.
Project description:Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt-Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT-TTR and L55P-TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P-TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single alpha-helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation-prone conformations characterized by full displacement of strands C and D from the main beta-sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H-bond network and consequent destabilization of the CBEF beta-sheet of the beta-sandwich; (v) WT forms aggregation-compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P-TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.
Project description:Liquid cellular compartments form in the cyto- or nucleoplasm and can regulate aberrant protein aggregation. Yet, the mechanisms by which these compartments affect protein aggregation remain unknown. Here, we combine kinetic theory of protein aggregation and liquid-liquid phase separation to study the spatial control of irreversible protein aggregation in the presence of liquid compartments. We find that even for weak interactions aggregates strongly partition into the liquid compartment. Aggregate partitioning is caused by a positive feedback mechanism of aggregate nucleation and growth driven by a flux maintaining the phase equilibrium between the compartment and its surrounding. Our model establishes a link between specific aggregating systems and the physical conditions maximizing aggregate partitioning into the compartment. The underlying mechanism of aggregate partitioning could be used to confine cytotoxic protein aggregates inside droplet-like compartments but may also represent a common mechanism to spatially control irreversible chemical reactions in general.
Project description:Systemic amyloidoses result from the aberrant secretion of destabilized, amyloidogenic proteins to the serum where they aggregate into proteotoxic soluble aggregates and amyloid fibrils. Few therapeutic approaches exist to attenuate extracellular pathologic aggregation of amyloidogenic proteins, necessitating the development of new strategies to intervene in these devastating disorders. We show that stress-independent activation of the Unfolded Protein Response-associated transcription factor ATF6 increases ER quality control stringency for the amyloidogenic protein transthyretin (TTR), preferentially reducing secretion of disease-associated TTR variants to an extent corresponding to the variants' destabilization of the TTR tetramer. This decrease in destabilized TTR variant secretion attenuates extracellular, concentration-dependent aggregation of amyloidogenic TTRs into soluble aggregates commonly associated with proteotoxicity in disease. Collectively, our results indicate that increasing ER quality control stringency through ATF6 activation is a strategy to attenuate pathologic aggregation of a destabilized, amyloidogenic protein, revealing a potential approach to intervene in systemic amyloid disease pathology.
Project description:Aggregation of ?-amyloid (A?) is widely believed to cause neuronal dysfunction in Alzheimer's disease. Transthyretin (TTR) binds to A? and inhibits its aggregation and neurotoxicity. TTR is a homotetrameric protein, with each monomer containing a short ?-helix and two anti-parallel ?-sheets. Dimers pack into tetramers to form a hydrophobic cavity. Here we report the discovery of a TTR mutant, N98A, that was more effective at inhibiting A? aggregation than wild-type (WT) TTR, although N98A and WT bound A? equally. The N98A mutation is located on a flexible loop distant from the putative A?-binding sites and does not alter secondary and tertiary structures nor prevent correct assembly into tetramers. Under non-physiological conditions, N98A tetramers were kinetically and thermodynamically less stable than WT, suggesting a difference in the tetramer folded structure. In vivo, the lone cysteine in TTR is frequently modified by S-cysteinylation or S-sulfonation. Like the N98A mutation, S-cysteinylation of TTR modestly decreased tetramer stability and increased TTR's effectiveness at inhibiting A? aggregation. Collectively, these data indicate that a subtle change in TTR tetramer structure measurably increases TTR's ability to inhibit A? aggregation.
Project description:Pathway complexity in supramolecular polymerization has recently sparked interest as a method to generate complex material behavior. The response of these systems relies on the existence of a metastable, kinetically trapped state. In this work, we show that strong switch-like behavior in supramolecular polymers can also be achieved through the introduction of competing aggregation pathways. This behavior is illustrated with the supramolecular polymerization of a porphyrin-based monomer at various concentrations, solvent compositions, and temperatures. It is found that the monomers aggregate via an isodesmic mechanism in weakly coupled J-type aggregates at intermediate solvent quality and temperature, followed by nucleated H-aggregates at lower solvent qualities and temperatures. At further increased thermodynamic driving forces, such as high concentration and low temperature, the H-aggregates can form hierarchical superhelices. Our mathematical models show that, contrary to a single-pathway polymerization, the existence of the isodesmic aggregation pathway buffers the free monomer pool and renders the nucleation of the H-aggregates insensitive to concentration changes in the limit of high concentrations. We also show that, at a given temperature or solvent quality, the thermodynamically stable aggregate morphology can be selected by controlling the remaining free external parameter. As a result, the judicious application of pathway complexity allows us to synthesize a diverse set of materials from only a single monomer. We envision that the engineering of competing pathways can increase the robustness in a wide variety of supramolecular polymer materials and lead to increasingly versatile applications.
Project description:Transthyretin (TTR) amyloidosis is associated with tissue deposition of TTR aggregates. TTR aggregation is initiated by dissociation of the native tetramer to form a monomeric intermediate, which locally unfolds and assembles into soluble oligomers and higher-order aggregates. However, a detailed mechanistic understanding requires kinetic and structural characterization of the low population intermediates formed. Here, we show that the monomeric intermediate exchanges with an ensemble of oligomers on the millisecond timescale. This transient and reversible exchange causes broadening of the <sup>19</sup>F resonance of a trifluoromethyl probe coupled to the monomeric intermediate at S85C. We show the <sup>19</sup>F linewidth and R<sub>2</sub> relaxation rate increase with increasing concentration of the oligomer. Furthermore, introduction of <sup>19</sup>F probes at additional TTR sites yielded distinct <sup>19</sup>F chemical shifts for the TTR tetramer and monomer when the trifluoromethyl probe was attached at S100C, located near the same subunit interface as S85C, but not with probes attached at S46C or E63C, which are distant from any interfaces. The <sup>19</sup>F probe at E63C shows that part of the DE loop, which is solvent accessible in the tetramer, becomes more buried in the NMR-visible oligomers. Finally, using backbone amides as probes, we show that parts of the EF helix and H-strand become highly flexible in the otherwise structured monomeric intermediate at acidic pH. We further find that TTR aggregation can be reversed by increasing pH. Taken together, this work provides insights into location-dependent conformational changes in the reversible early steps of a kinetically concerted TTR aggregation pathway.