The Coupling Between Folding, Zinc Binding, and Disulfide Bond Status of Human Cu, Zn Superoxide Dismutase: A Dissertation

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were \u3e50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate

Luminidependens (LD) is an Arabidopsis protein with prion behavior

Prion proteins provide a unique mode of biochemical memory through self-perpetuating changes in protein conformation and function. They have been studied in fungi and mammals, but not yet identified in plants. Using a computational model, we identified candidate prion domains (PrDs) in nearly 500 plant proteins. Plant flowering is of particular interest with respect to biological memory, because its regulation involves remembering and integrating previously experienced environmental conditions. We investigated the prion-forming capacity of three prion candidates involved in flowering using a yeast model, where prion attributes are well defined and readily tested. In yeast, prions heritably change protein functions by templating monomers into higher-order assemblies. For most yeast prions, the capacity to convert into a prion resides in a distinct prion domain. Thus, new prion-forming domains can be identified by functional complementation of a known prion domain. The prion-like domains (PrDs) of all three of the tested proteins formed higher-order oligomers. Uniquely, the Luminidependens PrD (LDPrD) fully replaced the prion-domain functions of a well-characterized yeast prion, Sup35. Our results suggest that prion-like conformational switches are evolutionarily conserved and might function in a wide variety of normal biological processes.Howard Hughes Medical InstituteG. Harold and Leila Y. Mathers FoundationEleanor Schwartz Charitable FoundationNational Science Foundation (U.S.). Graduate Research Fellowship Progra

Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1

Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Grant GM25874)National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant 1122374)G. Harold and Leila Y. Mathers FoundationBeckman Laser Institute FoundationEleanor Schwartz Charitable FoundationWhitehead Institute for Biomedical Researc

Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals

Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species.

Cu, Zn superoxide dismutase (SOD1) is a dimeric metal binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are \u3e95% folded at neutral pH and 37 degrees C, A4V, L38V, G93A, and L106V ranged from 50% to ~90% unfolded. The reduction of the disulfide-bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS-variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the stable native dimeric state

Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase.

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The approximately 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this beta-barrel protein

Microsecond hydrophobic collapse in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein

Using small-angle X-ray scattering combined with a continuous-flow mixing device, we monitored the microsecond compaction dynamics in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein. A significant collapse of the radius of gyration from 30 A to 23.2 A occurs within 300 micros after the initiation of refolding by a urea dilution jump. The subsequent folding after the major chain collapse occurs on a considerably longer time-scale. The protein folding trajectories constructed by comparing the development of the compactness and the secondary structure suggest that the specific hydrophobic collapse model rather than the framework model better explains the experimental observations. The folding trajectory of this alpha/beta-type protein is located between those of alpha-helical and beta-sheet proteins, suggesting that native structure determines the folding landscape