Early steps in oxidation-induced SOD1 misfolding: implications for non-amyloid protein aggregation in familial ALS.

Among the diseases of protein misfolding, amyotrophic lateral sclerosis (ALS) is unusual in that the proteinaceous neuronal inclusions that are the hallmark of the disease have neither the classic fibrillar appearance of amyloid by transmission electron microscopy nor the affinity for the dye Congo red that is a defining feature of amyloid. Mutations in the Cu, Zn superoxide dismutase (SOD1) cause the largest subset of inherited ALS cases. The mechanism by which this highly stable enzyme misfolds to form non-amyloid aggregates is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that SOD1's normal free radical scavenger role puts it at risk of oxidative damage and that it is this damage that triggers the misfolding primed by mutation. Here, we present evidence that hydrogen peroxide treatment, which generates free radical species at the SOD1 active site, causes oxidative damage to active-site histidine residues, leading to major structural changes and non-amyloid aggregation similar to that seen in ALS. Time-resolved measurements of release of bound metal ligands, exposure of hydrophobic surface area, and alterations in the SOD1 proton NMR spectrum have allowed us to model the early structural changes occurring as SOD1 misfolds, prior to aggregation. ALS-causing SOD1 mutations apparently alter this pathway by increasing exposure of buried epitopes in misfolded species populated at endpoint. We have identified a well-populated early misfolding intermediate that could serve as a target for therapies designed to block downstream misfolding and aggregation events and thereby treat SOD1-associated ALS.

Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form.

Amyotrophic lateral sclerosis (ALS) is a conformational disease in which misfolding and aggregation of proteins such as SOD1 (familial ALS) and TDP-43 (sporadic ALS) are central features. The conformations adopted by such proteins within motor neurons in affected patients are not well known. We have developed a novel conformation-specific antibody (USOD) targeted against SOD1 residues 42-48 that specifically recognizes SOD1 in which the beta barrel is unfolded. Use of this antibody, in conjunction with the previously described SEDI antibody that recognizes the SOD1 dimer interface, allows a detailed investigation of the in vivo conformation of SOD1 at the residue-specific level. USOD and SEDI immunohistochemistry of spinal cord sections from ALS cases resulting from SOD1 mutations (A4V and DeltaG27/P28) shows that inclusions within remaining motor neurons contain SOD1 with both an unfolded beta barrel and a disrupted dimer interface. Misfolded SOD1 can also be immunoprecipitated from spinal cord extracts of these cases using USOD. However, in ten cases of sporadic ALS, misfolded SOD1 is not detected by either immunohistochemistry or immunoprecipitation. Using the amyloid-specific dyes, Congo Red and Thioflavin S, we find that SOD1-positive inclusions in familial ALS, as well as TDP-43- and ubiquitin-positive inclusions in sporadic ALS, contain non-amyloid protein deposits. We conclude that SOD1 misfolding is not a feature of sporadic ALS, and that both SOD1-ALS and sporadic ALS, rather than being amyloid diseases, are conformational diseases that involve amorphous aggregation of misfolded protein. This knowledge will provide new insights into subcellular events that cause misfolding, aggregation and toxicity.

Denaturational stress induces formation of zinc-deficient monomers of Cu,Zn superoxide dismutase: implications for pathogenesis in amyotrophic lateral sclerosis.

Mutations in the Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis cases. SOD1 is a homodimer in which each monomer binds one copper atom and one zinc atom. Mutation is believed to increase the conformational flexibility of SOD1, giving rise to a misfolded SOD1 population with novel cytotoxic properties. While SOD1's metal ligands affect its stability greatly, little is known about the role these metals play in the folding, unfolding, and misfolding processes. Here, we present a method by which we were able to measure the rates of metal release during SOD1 unfolding in guanidine hydrochloride. Rates of dimer dissociation, measured by a time-resolved cross-linking assay, and conformational changes in SOD1's beta-barrel core, monitored by tryptophan fluorescence intensity, were compared with the rates of copper release and zinc release. Correlations were observed across a range of denaturant concentrations, giving rise to a more detailed model of the SOD1 unfolding process than was previously available. According to this model, the major unfolding pathway involves simultaneous dimer dissociation and zinc release as an early step that is followed by a slow conformational change in the protein's core, which, in turn, is followed by rapid copper release. This model establishes a zinc-deficient, copper-loaded SOD1 monomer as a well-populated SOD1 unfolding intermediate and a species likely to be populated under conditions of denaturational stress. Because the cytotoxicity of zinc-deficient SOD1 has been demonstrated previously, this species is a good candidate for the cytotoxic species in SOD1-associated amyotrophic lateral sclerosis.

Conformation of a double-membrane-spanning fragment of a G protein-coupled receptor: effects of hydrophobic environment and pH.

Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133-140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the mu-opioid receptor ('TM2-3') that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2-3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the mu-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2-3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix-helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2-3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2-3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.

Role of metal ions in ligand-receptor interaction: insights from structural studies.

Experimental data indicate that metal ions such as Na(+), Ca(2+) and Mg(2+), which are present in millimolar concentrations in the extracellular environment, modulate binding of ligands to plasma membrane receptors. Here, we briefly review structural studies that demonstrate that various types of ligands, including peptide hormones and drugs, bind metal ions, in particular Ca(2+), in the lipid milieu. We propose that the metal ion-bound forms of ligands represent their bioactive conformations. With a view to understanding the mechanism of modulation of ligand-receptor interactions by metal ions, we have computed a homology model of the mu-opioid receptor, a G protein-coupled receptor (GPCR), and performed docking of specific agonist and antagonist ligands in the receptor. This resulted in the formation of a ligand-metal ion-receptor (ternary) complex which accounted for the data on the structure-activity relationships of ligands and mutation data on the receptor. Based on experimental and modeling data, we have proposed a general mechanism of activation of GPCRs by their corresponding ligands wherein metal ions play a pivotal role. Studies on overexpressed segments of mu-receptor are in progress to verify the above proposal.

Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor.

We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human mu-opioid receptor. The fragment ('TM2-3'), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2-3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2-3 in lysophosphatidylcholine micelles showed that TM2-3 adopts approximately 50% alpha-helical structure in this environment, with the remainder consisting of disordered and/or beta-structure. This is consistent with the assumption of an alpha-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2-3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the mu-opioid receptor and, possibly, other G-protein-coupled receptors as well.