Cu/Zn Superoxide Dismutase Misfolding in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration resulting in progressive paralysis and death. The only known cause of typical ALS is mutations in SOD1; these predominantly missense mutations produce a toxic gain-of-function in the enzyme Cu/Zn superoxide dismutase (SOD1). The prevailing hypotheses regarding the mechanism of toxicity were a) oxidative damage from aberrant SOD1 redox chemistry, and b) misfolding of the mutant protein. The goal of this thesis was to investigate the molecular mechanisms of the mutant SOD1 (mSOD1) misfolding and toxicity. We proposed that oxidative damage to SOD1 itself could cause its misfolding and aggregation. To investigate this hypothesis, we subjected purified SOD1 in vitro to metal catalyzed oxidation. Oxidation of SOD1 produced aggregates reminiscent of those observed in ALS pathology. Aggregation propensity of zinc-deficient SOD1 and several mSOD1s known to have lower zinc-binding affinity was proportional to partial unfolding. Oxidation of SOD1 caused conversion of several His residues to 2-oxo-histidine. Because oxidation of SOD1 primarily affected the metal-binding His residues, we hypothesized that oxidation of wild-type, holo-SOD1 should lead to aggregation. Increasing the concentration of wild-type SOD1 in oxidation reactions produced aggregates similar to those observed earlier. Both wild-type and mSOD1 aggregation kinetics revealed an initial decrease in particle size rather than a monotonic increase using dynamic light scattering. This was consistent with the conversion of SOD1, normally an obligate homodimer, into monomers prior to aggregation. This observation was confirmed using analytical ultracentrifugation. The common aggregation pathway for wild-type and mSOD1 suggested a mechanism for sporadic ALS caused by SOD1 misfolding. To interrogate the in vivo misfolding pathway of SOD1, we used its high-resolution structure to create an antibody that reacts with monomer/misfolded SOD1 but not the native dimer. Upon verifying the reactivity of this antibody, we showed that monomer/misfolded SOD1 is found in a human case of familial ALS and in transgenic animal models of ALS. Misfolded SOD1 is found primarily in affected cells, motor neurons. Misfolded SOD1 is also initially absent, but appears prior to symptom onset. These observations together suggest a causal role for SOD1 misfolding through a monomeric intermediate in ALS pathogenesis.Ph

Structure, folding, and misfolding of Cu,Zn superoxide dismutase in amyotrophic lateral sclerosis

AbstractFourteen years after the discovery that mutations in Cu, Zn superoxide dismutase (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS), the mechanism by which mutant SOD1 exerts toxicity remains unknown. The two principle hypotheses are (a) oxidative damage stemming from aberrant SOD1 redox chemistry, and (b) misfolding of the mutant protein. Here we review the structure and function of wild-type SOD1, as well as the changes to the structure and function in mutant SOD1. The relative merits of the two hypotheses are compared and a common unifying principle is outlined. Lastly, the potential for therapies targeting SOD1 misfolding is discussed

A Novel Destabilizing Domain Based on a Small-Molecule Dependent Fluorophore

Tools that can directly regulate the activity of any protein-of-interest are valuable in the study of complex biological processes. Herein, we describe the development of a novel protein domain that exhibits small molecule-dependent stability and fluorescence based on the bilirubin-inducible fluorescent protein, UnaG. When genetically fused to any protein-of-interest, this fluorescent destabilizing domain (FDD) confers its instability to the entire fusion protein, facilitating the rapid degradation of the fusion. In the presence of its cognate ligand bilirubin (BR), the FDD fusion becomes stable and fluorescent. This new chemical genetic tool allows for rapid, reversible, and tunable control over the stability and fluorescence of a wide range of protein targets

Intracellular Context Affects Levels of a Chemically Dependent Destabilizing Domain

<div><p>The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.</p> </div

General Method for Regulating Protein Stability with Light

Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination

ER and secreted destabilizing domains.

<p>(A) Fluorescence micrographs of eDD cells. The overlay image shows eDD (green), ER-Tracker Red (red), and Hoechst stain (blue). (B) Flow cytometry of eDD cells with Shield-1 (2 µM) or vehicle control after a 6 hour incubation. Cells were co-treated with cycloheximide (CHX, 5 µg/mL), MG132 (5 µM), or brefeldin-A (BFA, 2.5 µg/mL). * P-value<0.05, ** P-value<0.005. (C) Bioluminescence quantification of media from eDDs cells after exposure to vehicle control, Shield-1 (1 µM), CHX (1 µg/mL), or co-treatment with both Shield-1 and CHX. (D) Bioluminescence quantification of media from eDDs cells after exposure to vehicle control, Shield-1 (1 µM), MG132 (1 µM), or co-treatment with both. Error bars represent ± S.E.M. (n = 3).</p

N- and C-terminal DDs targeted to the mitochondria.

<p>(A) Fluorescence micrographs of mDDn cells in the presence and absence of Shield-1 (1 µM). The overlay image shows mDD (XFP, green), Mitotracker Orange (red), and Hoechst stain (blue). (B) Fluorescence micrographs of mDDc cells as in (A). (C) Flow cytometry of mDDn cells with Shield-1 (2 µM) or vehicle control after a 6 hour incubation. Cells were co-treated with cycloheximide (CHX, 5 µg/mL), MG132 (5 µM), or both CHX and MG132. (D) Flow cytometry of mDDn cells exposed to small molecules as in (C). Micrograph scale bars indicate 10 microns. Error bars represent ± S.E.M. (n = 3).</p

Substoichiometric inhibition of transthyretin misfolding by immune-targeting sparsely populated misfolding intermediates : a potential diagnostic and therapeutic for TTR amyloidoses

Wild-type and mutant transthyretin (TTR) can misfold and deposit in the heart, peripheral nerves, and other sites causing amyloid disease. Pharmacological chaperones, Tafamidis (R) and diflunisal, inhibit TTR misfolding by stabilizing native tetrameric TTR; however, their minimal effective concentration is in the micromolar range. By immune-targeting sparsely populated TTR misfolding intermediates (i.e. monomers), we achieved fibril inhibition at substoichiometric concentrations. We developed an antibody (misTTR) that targets TTR residues 89-97, an epitope buried in the tetramer but exposed in the monomer. Nanomolar misTTR inhibits fibrillogenesis of misfolded TTR under micromolar concentrations. Pan-specific TTR antibodies do not possess such fibril inhibiting properties. We show that selective targeting of misfolding intermediates is an alternative to native state stabilization and requires substoichiometric concentrations. MisTTR or its derivative may have both diagnostic and therapeutic potential