Electrostatic control of Cu, Zn superoxide dismutase aggregation in Amyotrophic Lateral Sclerosis : from lysine modification to interaction with lipid membranes.

Abstract

Cu, Zn superoxide dismutase (SOD1) is a ubiquitous metalloprotein, which is responsible for protecting living cells from oxidative stress via disproportionation of superoxide ions. Misfolding and subsequent aggregation of SOD1 is casually linked to familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Symptoms of ALS included weakness in arms and legs and respiratory failure, which leads to death within a few years. The deposition of SOD1 amyloid fibrils (i.e., gain of toxic function) in axons leads to selective death of motor neurons and neurodegeneration. In this dissertation, I use various bioanalytical and biophysical techniques to study aggregation of wild-type (WT) and ALS-variant apo-SOD1 in both in vitro and in vivo conditions, in the context of ALS. In Chapter Two of this dissertation, I first show how acylation of lysine residues in WT apo-SOD1 affects the kinetics and morphology of amyloid formation during microplate-based (in vitro) assays. Moreover, the ability of WT SOD1 fibrils to seed the aggregation of ALS-variant SOD1 in organotypic spinal cord derived from transgenic YFP-G85R-SOD1 mice is studied. I show, for the first time, that minimal acylation of apo-SOD1 with small moieties carrying a high negative charge increases the probability of formation of non-toxic SOD1 oligomers. The results from this study are crucial for designing small molecules that can selectively bind to and inhibit the propagation of toxic SOD1 fibrils. In Chapter Three of this dissertation, I determine the rate, mechanism of formation, and morphology of aggregated forms of WT apo-SOD1 in the presence of small unilamellar vesicles (SUVs) composed of identical hydrophobic chains, but headgroups of variable charge i.e., anionic, zwitterionic, and cationic. The aggregation of eight different ALS variants of apo-SOD1 was also examined in the presence of SUVs. I found that SOD1 aggregation can be triggered by negatively-charged liposomes, which is very significant considering that cell-to-cell propagation of SOD1 is mediated by its interaction with lipid membranes.