Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction

Tiwari, Ashutosh; Hayward, Lawrence J.

doi:10.1074/jbc.m210419200

Cited by 202 publications

(216 citation statements)

References 66 publications

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“…Metal-free forms of SOD1 have been proposed to be the toxic precursor preceding aggregation in familial aLS [43,44]. the majority of aLS mutations affect the structural stability of SOD1, leading to the exposure of buried hydrophobic residues, thus resulting in aggregation [45]. hence SOD1 Wt has an intrinsic propensity to aggregate which is enhanced by mutation.…”

Section: Discussionmentioning

confidence: 99%

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Sundaramoorthy

Walker

Yerbury

et al. 2013

Cell. Mol. Life Sci.

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Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of ALS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. ALS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. However, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of ALS pathology. Similarly, whilst dysfunction to the ERGolgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial ALS, it remains unclear whether misfolded, wildtype SOD1 triggers ER-Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. Hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the ER-Golgi apparatus, leading to Golgi fragmentation, induction of ER stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces ER-Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. Hence extracellular misfolded wildtype or mutant SOD1 induce dysfunction to ERGolgi compartments characteristic of ALS in neuronal cells, implicating extracellular SOD1 in the spread of pathology among motor neurons in both sporadic and familial ALS. Abstract amyotrophic lateral sclerosis (aLS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of aLS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. aLS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. however, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of aLS pathology. Similarly, whilst dysfunction to the eR-Golgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial aLS, it remains unclear whether misfolded, wildtype SOD1 triggers eR-Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the eR-Golgi apparatus, leading to Golgi fragmentation, induction of eR stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces eR-Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. hence extracellu...

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Section: Discussionmentioning

confidence: 99%

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Sundaramoorthy

Walker

Yerbury

et al. 2013

Cell. Mol. Life Sci.

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“…9). Several papers (41,46) have also reported that H46R is less stable than other FALS mutants in vitro. Most interestingly, the immunoreactivities of H46R against the mAbs in Western blot and ELISA were similar to that of WT or C111S (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…9C). Tiwari and Hayword (41) reported that, in the presence of disulfide-reducing agents, FALS mutant SODs were more susceptible than WT to aberrant migration during partially denaturing SDS-PAGE and to proteolytic digestion. A conserved internal disulfide bond between Cys-57 and Cys-146 also contributes to the stability of Cu/Zn-SOD.…”

Section: Discussionmentioning

confidence: 99%

Different Immunoreactivity against Monoclonal Antibodies between Wild-type and Mutant Copper/Zinc Superoxide Dismutase Linked to Amyotrophic Lateral Sclerosis

Fujiwara

Miyamoto

Ogasahara

et al. 2005

Journal of Biological Chemistry

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show less enzymatic activities, many retain full activity (4). Several lines of transgenic mice that express mutant human SOD1 linked with FALS developed progressive neurodegeneration and a phenotype that clearly resembles human FALS (5, 6) despite having higher SOD activity. In contrast, SOD1-knock out mice do not exhibit motor neuron dysfunction (7). These findings suggest that this disease is the result of a toxic gain of function but not a loss in SOD activity. Several different hypotheses have been proposed to explain the toxic gain of function, including the production of ROS, mitochondrial defects, glutamate-induced excitotoxicity, neurofilament inclusions, and the formation of intracellular toxic protein aggregates (reviewed in Ref. 8). However, the mechanism by which FALS mutant SODs causes motor neuron degeneration is not completely understood.The tertiary structure of Cu/Zn-SOD is characterized by the presence of a Greek key ␤-barrel containing an internal disulfide bond between Cys-57 and Cys-146 (9, 10), both of which contribute to its high stability. On the other hand, various FALS mutant SODs show a decreased stability and a lower level of metallation (4,11,12). Several reports, including ours ( Fig. 1), 2 reported that FALS mutant SODs exhibit an accelerated turnover or an increased susceptibility to degradation in proteasome (13,14) and suggest that unfolding or conformational perturbations of mutant SOD proteins occur in vivo. Niwa et al. (15) reported that Dorfin, a RING finger-type ubiquitin-protein isopeptide ligase (E3) ubiqutinates the FALS mutant SODs but not wild-type Cu/Zn-SOD, suggesting that FALS mutant SODs have a unique structure that can be recognized by Dorfin. We reported previously that FALS mutant SODs are more susceptible to glycation or fructation (16) and that they form aggregates at a higher rate than the wild-type SOD, when incubated in the presence of copper ions (4).

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“…These SOD1 multimers could be involved in protein aggregation and the pathology of amyotrophic lateral sclerosis. Interestingly, increased susceptibility to disulfide reduction has been observed in some fALS mutants (36); therefore, protein monomerization caused by the disulfide reduction and demetallation might be an important process in causing the fALS diseases.…”

Section: Discussionmentioning

confidence: 99%

The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status

Arnesano

Banci

Bertini

et al. 2004

Journal of Biological Chemistry

231

234

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The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-todimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any posttranslational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis.Eukaryotic copper,zinc superoxide dismutase (SOD1) 1 catalyzes the dismutation of superoxide radical to oxygen and hydrogen peroxide and is a 32-kDa homodimeric enzyme found predominantly in the cytosol (1). SOD1 is one of the most thermally stable enzymes known in mesophilic organisms. Dismutase activity declines at 80°C with a corresponding melting temperature, T m , above 90°C (2). The protein is stable in the presence of strong denaturants, and the activity is observed in 4% SDS or 10 M urea (3). Structural properties of SOD1 that contribute to this extreme thermochemical stability are thought to include an eight-stranded ␤-barrel motif, hydrophobic interactions associated with dimerization, coordinate covalent bonds, and an intrasubunit disulfide bond between highly conserved pair of cysteines, namely Cys 57 and Cys 146 in the human form. Whereas the dimerization can contribute to the structural stability through the reduction of its mobility (4), the roles of the disulfide bond in the SOD1 function and/or structure are only now beginning to emerge. Inspection of the SOD1 structure reveals that the loop containing Cys 57 can influence the conformation of the catalytically important residue, Arg 143 , through a hydrogen-bonding network (5). Portions of this loop contribute to the dimer interface (6), leading to the possibility that the disulfide bond influences the protein dimerization and thereby the SOD1 quaternary structure.To attain th...

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Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction

Cited by 202 publications

References 66 publications

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Different Immunoreactivity against Monoclonal Antibodies between Wild-type and Mutant Copper/Zinc Superoxide Dismutase Linked to Amyotrophic Lateral Sclerosis

The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status

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