Evan T. Powers

Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade.

The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A–retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (Kds ∼2 nM and ∼200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity.

Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease; however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V122I), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V122I TTR have nearly equivalent tetramer stabilities, V122I cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance—presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30M/T119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.

Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss

Significance Communication between nerve cells occurs at specialized cellular structures known as synapses. Loss of synaptic function is associated with cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here we describe a pathway for synaptic damage whereby amyloid-β1–42 peptide (Aβ1–42) releases, via stimulation of α7 nicotinic receptors, excessive amounts of glutamate from astrocytes, in turn activating extrasynaptic NMDA-type glutamate receptors (eNMDARs) to mediate synaptic damage. The Food and Drug Administration-approved drug memantine offers some beneficial effect, but the improved eNMDAR antagonist NitroMemantine completely ameliorates Aβ-induced synaptic loss, providing hope for disease-modifying intervention in AD. Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aβ induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aβ-induced damage both in vitro and in vivo.

Context-dependent contributions of backbone hydrogen bonding to β-sheet folding energetics

Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small β-sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the proteins loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a β-sheet protein.

Amyloid-β forms fibrils by nucleated conformational conversion of oligomers

Aβ amyloidogenesis is reported to occur via a nucleated polymerization mechanism, if so the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many laboratories have detected early non-fibrillar Aβ oligomers without observing amyloid fibrils, suggesting a mechanistic revision may be needed. Herein, we introduce Cys-Cys-Aβ1-40 that cannot bind to the latent fluorophore FlAsH as a monomer, but is capable of binding FlAsH as an non-fibrillar oligomer or as a fibril, rendering the conjugates fluorescent. FlAsH monitoring of Cys-Cys-Aβ1-40 aggregation provides compelling evidence that Aβ1-40 very rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism (seedable). Moreover, this methodology demonstrated that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-associated oligomerization phase of Aβ amyloidogenesis, while allowing fibril formation, rationalizing how low plasmalogen ethanolamine levels in the brain are epidemiologically linked to Alzheimer’s disease.

ALS Mutants of Human Superoxide Dismutase Form Fibrous Aggregates Via Framework Destabilization

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.

The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability

The folding energetics of the mono-N-glycosylated adhesion domain of the human immune cell receptor cluster of differentiation 2 (hCD2ad) were studied systematically to understand the influence of the N-glycan on the folding energy landscape. Fully elaborated N-glycan structures accelerate folding by 4-fold and stabilize the β-sandwich structure by 3.1 kcal/mol, relative to the nonglycosylated protein. The N-glycans first saccharide unit accounts for the entire acceleration of folding and for 2/3 of the native state stabilization. The remaining third of the stabilization is derived from the next 2 saccharide units. Thus, the conserved N-linked triose core, ManGlcNAc2, improves both the kinetics and the thermodynamics of protein folding. The native state stabilization and decreased activation barrier for folding conferred by N-glycosylation provide a powerful and potentially general mechanism for enhancing folding in the secretory pathway.

An Adaptable Standard for Protein Export from the Endoplasmic Reticulum

To provide an integrated view of endoplasmic reticulum (ER) function in protein export, we have described the interdependence of protein folding energetics and the adaptable biology of cellular protein folding and transport through the exocytic pathway. A simplified treatment of the protein homeostasis network and a formalism for how this network of competing pathways interprets protein folding kinetics and thermodynamics provides a framework for understanding cellular protein trafficking. We illustrate how folding and misfolding energetics, in concert with the adjustable biological capacities of the folding, degradation, and export pathways, collectively dictate an adaptable standard for protein export from the ER. A model of folding for export (FoldEx) establishes that no single feature dictates folding and transport efficiency. Instead, a network view provides insight into the basis for cellular diversity, disease origins, and protein homeostasis, and predicts strategies for restoring protein homeostasis in protein-misfolding diseases.

Diversity in the origins of proteostasis networks- a driver for protein function in evolution

Although the sequence of a protein largely determines its function, proteins can adopt different folding states in response to changes in the environment, some of which may be deleterious to the organism. All organisms —Bacteria, Archaea and Eukarya — have evolved a protein homeostasis, or proteostasis, network comprising chaperones and folding factors, degradation components, signalling pathways and specialized compartmentalized modules that manage protein folding in response to environmental stimuli and variation. Surveying the origins of proteostasis networks reveals that they have co-evolved with the proteome to regulate the physiological state of the cell, reflecting the unique stresses that different cells or organisms experience, and that they have a key role in driving evolution by closely managing the link between the phenotype and the genotype.

Localized thermodynamic coupling between hydrogen bonding and microenvironment polarity substantially stabilizes proteins

The energetic contributions of hydrogen bonding to protein folding are still unclear, despite more than 70 years of study. This is due partly to the difficulty of extracting thermodynamic information about specific interactions from protein mutagenesis data and partly to the context dependence of hydrogen bond strengths. Herein, we test the hypothesis that hydrogen bond strengths depend on the polarity of their microenvironment, with stronger hydrogen bonds forming in nonpolar surroundings. Double-mutant cycle analysis using a combination of amide-to-ester backbone mutagenesis and traditional side chain mutagenesis revealed that hydrogen bonds can be stronger by up to 1.2 kcal mol−1 when they are sequestered in hydrophobic surroundings than when they are solvent exposed. Such large coupling energies between hydrogen bond strengths and local polarity suggest that the context dependence of hydrogen bond strengths must be accounted for in any comprehensive account of the forces responsible for protein folding.