Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease afflicting the voluntary motor system. More than 100 different mutations in the ubiquitously expressed enzyme superoxide dismutase-1 (SOD1) have been associated with the disease. To search for the nature of the cytotoxicity of mutant SOD1s, amounts, enzymic activities and structural properties of the protein as well as the CNS histopathology were examined in multiple transgenic murine models. In order to generate the ALS phenotype within the short lifespan of the mouse, more than 20-fold increased rates of synthesis of mutant SOD1s appear to be required. The organs of transgenic mice expressing human wild-type SOD1 or either of the G93A and D90A mutant proteins showed high steady-state protein levels. The major proportion of these SOD1s in the CNS were inactive due to insufficient Cu charging and all contained subfractions with a reduced C57-C146 intrasubunit disulphide bond. Both G85R and the truncated G127insTGGG mutant showed low steady-state protein levels, lacked enzyme activity and had no C57-C146 disulphide bond. These mutants were also enriched in the CNS relative to other organs, suggesting inefficient recognition and degradation of misfolded disulphide-reduced SOD1 in susceptible tissues. In end-stage disease, despite 35-fold differences in levels of mutant SOD1s, similar amounts of detergent-resistant aggregates accumulated in the spinal cord. Small granular as well as larger more diffuse human SOD1 (hSOD1)-inclusions developed in all strains, the latter more pronounced in those with high hSOD1 levels. Widespread vacuolizations were seen in the strains with high levels of hSOD1 but not those with low, suggesting these alterations to be artefacts related to high hSOD1 levels and not to the ALS-causing cytotoxicity. The findings suggest that the motoneuron degeneration could be due to long-term exposure to misfolded aggregation-prone disulphide-reduced SOD1, which constitutes minute subfractions of the stable mutants and larger proportions of the unstable mutants.

The molecular mechanism by which the homodimeric enzyme Cu͞Zn superoxide dismutase (SOD) causes neural damage in amytrophic lateral sclerosis is yet poorly understood. A striking, as well as an unusual, feature of SOD is that it maintains intrasubunit disulfide bonds in the reducing environment of the cytosol. Here, we investigate the role of these disulfide bonds in folding and assembly of the SOD apo protein (apoSOD) homodimer through extensive protein engineering. The results show that apoSOD folds in a simple three-state process by means of two kinetic barriers: 2Dº2MºM 2. The early predominant barrier represents folding of the monomers (M), and the late barrier the assembly of the dimer (M 2). Unique for this mechanism is a dependence of protein concentration on the unfolding rate constant under physiological conditions, which disappears above 6 M Urea where the transition state for unfolding shifts to first-order dissociation of the dimer in accordance with Hammond-postulate behavior. Although reduction of the intrasubunit disulfide bond C57-C146 is not critical for folding of the apoSOD monomer, it has a pronounced effect on its stability and abolishes subsequent dimerization. Thus, impaired ability to form, or retain, the C57-C146 bond in vivo is predicted to increase the cellular load of marginally stable apoSOD monomers, which may have implications for the amytrophic lateral sclerosis neuropathology.protein folding ͉ protein stability ͉ disulfide bond ͉ transition-state shifts ͉ protein engineering T he mechanism by which mutant superoxide dismutase (SOD) leads to neural damage in the familial form of amyotrophic lateral sclerosis (ALS) is yet unknown (1-3). In analogy with other neurodegenerative disorders (4), however, an increasing body of observations (5-16) suggest that the ALS disease mechanism is coupled to destabilization or misfolding of the SOD structure (17), manifested ultimately by cellular inclusions of SOD aggregates (3,18). From a strictly energetic perspective, the native SOD structure is distinctive by containing one oxidized disulfide bond per monomer (C57-C146) in the reducing environment of the cytosol (19) (Fig. 1). Normally, disulfide bonds are maintained only under oxidizing conditions in the extracellular space (20, 21) where they increase protein stability by confining the configurational entropy of the denatured ensemble (22). Indications that the integrity of the C57-C146 bond may have bearing on the molecular events in ALS were recently provided by Tiwari and Hayward (15), who demonstrated that ALS-associated SOD mutants are more susceptible to chemical disulfide reduction than the wild-type protein. More detailed elucidation of the structural and energetic effects accompanying mutational perturbations and disulfide reduction has so far been prevented by scarce knowledge about how the SOD homodimer folds (23).In this study, we shed further light on this issue by mapping out the folding and assembly reaction of metal-depleted SOD through kinetic and thermodynamic analysis of...

Amyotrophic lateral sclerosis is a neurodegenerative syndrome associated with 114 mutations in the gene encoding the cytosolic homodimeric enzyme Cu͞Zn superoxide dismutase (SOD). In this article, we report that amyotrophic lateral sclerosis-associated SOD mutations with distinctly different disease progression can be rationalized in terms of their folding patterns. The mutations are found to perturb the protein in multiple ways; they destabilize the precursor monomers (class 1), weaken the dimer interface (class 2), or both at the same time (class 1 ؉ 2). A shared feature of the mutational perturbations is a shift of the folding equilibrium toward poorly structured SOD monomers. We observed a link, coupled to the altered folding patterns, between protein stability, net charge, and survival time for the patients carrying the mutations.neurodegenerative disease ͉ protein stability

More than 100 point mutations of the superoxide scavenger Cu͞Zn superoxide dismutase (SOD; EC 1.15.1.1) have been associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, these mutations are scattered throughout the protein and provide no clear functional or structural clues to the underlying disease mechanism. Therefore, we undertook to look for foldingrelated defects by comparing the unfolding behavior of five ALS-associated mutants with distinct structural characteristics: A4V at the interface between the N and C termini, C6F in the hydrophobic core, D90A at the protein surface, and G93A and G93C, which decrease backbone flexibility. With the exception of the disruptive replacements A4V and C6F, the mutations only marginally affect the stability of the native protein, yet all mutants share a pronounced destabilization of the metal-free apo state: the higher the stability loss, the lower the mean survival time for ALS patients carrying the mutation. Thus organism-level pathology may be directly related to the properties of the immature state of a protein rather than to those of the native species.A myotrophic lateral sclerosis (ALS) is a motor neuron syndrome where a sub group of 3-6% has been associated with a diverse set of mutations in the superoxide scavenger Cu͞Zn superoxide dismutase (SOD; 1.15.1.1) (1, 2). Interestingly, several of these mutations show perfectly native-like in vivo activity (3, 4) and metal coordination (5). As an alternative cause of disease, transgenic mouse models suggest that ALS arises through an adverse, and yet-unidentified, side reaction of the mutated protein causing cytotoxicity. Mice devoid of SOD retain normal motor function (6), as do mice overexpressing wild-type SOD. In contrast, mice expressing high levels of mutant human SOD in addition to endogenous SOD show neural damage. The collective evidence suggests that mutant SOD has gained a cytopathogenic function (7,8). Models for how the neurodegenerating toxicity arise include the following: (i) enzymatic side-reactions in catalytically promiscuous SOD mutants producing increased levels of oxidative compounds such as hydroxyl radicals (9-11) and peroxynitrite (12), (ii) release of free Cu ions (13), (iii) aberrant binding of SOD to other proteins (14), (iv) binding of mutant SOD to heat shock proteins (15, 16) with the subsequent prevention of their antiapoptotic function (16), (v) altered redox regulation (17), and (vi) formation of toxic SOD aggregates (18)(19)(20). Evidence that could potentially distinguish between these models was recently provided from mice lacking the metal-loading chaperone CCS (copper chaperone for SOD; ref. 21). The chaperone is essential for incorporating the copper ion and, hence, to gain the native protein. When the CCS gene was ablated, the ALS-associated mutations were observed to still provoke the disease, indicating that a copper-free precursor state of SOD causes neurotoxicity (21). Along this line, a broad spectrum of mechanistic scenarios is implicated...

Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfA WT in complex only with DNA confirms that PrfA WT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.PrfA | glutathione | activation | Listeria | virulence