Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0002736 (amyotrophic lateral sclerosis)
 19,048 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations in Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS) through mechanisms proposed to involve SOD1 misfolding, but the intracellular factors that modulate folding and stability of SOD1 are largely unknown. By using yeast and mammalian expression systems, we demonstrate here that SOD1 stability is governed by post-translational modification factors that target the SOD1 disulfide. Oxidation of the human SOD1 disulfide in vivo was found to involve both the copper chaperone for SOD1 (CCS) and the CCS-independent pathway for copper activation. When both copper pathways were blocked, wild type SOD1 stably accumulated in yeast cells with a reduced disulfide, whereas ALS SOD1 mutants A4V, G93A, and G37R were degraded. We describe here an unprecedented role for the thiol oxidoreductase glutaredoxin in reducing the SOD1 disulfide and destabilizing ALS mutants. Specifically, the major cytosolic glutaredoxin of yeast was seen to reduce the intramolecular disulfide of ALS SOD1 mutant A4V SOD1 in vivo and in vitro. By comparison, glutaredoxin was less reactive toward the disulfide of wild type SOD1. The apo-form of A4V SOD1 was highly reactive with glutaredoxin but not SOD1 containing both copper and zinc. Glutaredoxin therefore preferentially targets the immature form of ALS mutant SOD1 lacking metal co-factors. Overall, these studies implicate a critical balance between cellular reductants such as glutaredoxin and copper activation pathways in controlling the disulfide and stability of SOD1 in vivo.
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PMID:The effects of glutaredoxin and copper activation pathways on the disulfide and stability of Cu,Zn superoxide dismutase. 1688 Feb 13

Converging evidence indicates that aberrant aggregation of mutant Cu,Zn-superoxide dismutase (mutSOD1) is strongly implicated in familial amyotrophic lateral sclerosis (FALS). MutSOD1 forms high molecular weight oligomers, which disappear under reducing conditions, both in neural tissues of FALS transgenic mice and in transfected cultured cells, indicating a role for aberrant intermolecular disulfide cross-linking in the oligomerization and aggregation process. To study the contribution of specific cysteines in the mechanism of aggregation, we mutated human SOD1 in each of its four cysteine residues and, using a cell transfection assay, analyzed the solubility and aggregation of those SOD1s. Our results suggest that the formation of mutSOD1 aggregates are the consequence of covalent disulfide cross-linking and non-covalent interactions. In particular, we found that the removal of Cys-111 strongly reduces the ability of a range of different FALS-associated mutSOD1s to form aggregates and impair cell viability in cultured NSC-34 cells. Moreover, the removal of Cys-111 impairs the ability of mutSOD1s to form disulfide cross-linking. Treatments that deplete the cellular pool of GSH exacerbate mutSOD1s insolubility, whereas an overload of intracellular GSH or overexpression of glutaredoxin-1, which specifically catalyzes the reduction of protein-SSG-mixed disulfides, significantly rescues mutSOD1s solubility. These data are consistent with the view that the redox environment influences the oligomerization/aggregation pathway of mutSOD1 and point to Cys-111 as a key mediator of this process.
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PMID:Cysteine 111 affects aggregation and cytotoxicity of mutant Cu,Zn-superoxide dismutase associated with familial amyotrophic lateral sclerosis. 1800 98

Vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS) arises from a combination of several mechanisms, including protein misfolding and aggregation, mitochondrial dysfunction and oxidative damage. Protein aggregates are found in motoneurons in models for ALS linked to a mutation in the gene coding for Cu,Zn superoxide dismutase (SOD1) and in ALS patients as well. Aggregation of mutant SOD1 in the cytoplasm and/or into mitochondria has been repeatedly proposed as a main culprit for the degeneration of motoneurons. It is, however, still debated whether SOD1 aggregates represent a cause, a correlate or a consequence of processes leading to cell death. We have exploited the ability of glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols either in the cytoplasm and in the IMS (Grx1) or in the mitochondrial matrix (Grx2) as a tool for restoring a correct redox environment and preventing the aggregation of mutant SOD1. Here we show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but does not inhibit mitochondrial damage and apoptosis induced by mutant SOD1 in neuronal cells (SH-SY5Y) or in immortalized motoneurons (NSC-34). Conversely, the overexpression of Grx2 increases the solubility of mutant SOD1 in mitochondria, interferes with mitochondrial fragmentation by modifying the expression pattern of proteins involved in mitochondrial dynamics, preserves mitochondrial function and strongly protects neuronal cells from apoptosis. The toxicity of mutant SOD1, therefore, mostly arises from mitochondrial dysfunction and rescue of mitochondrial damage may represent a promising therapeutic strategy.
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PMID:Glutaredoxin 2 prevents aggregation of mutant SOD1 in mitochondria and abolishes its toxicity. 2082 29

Selenium is an essential micronutrient required for cellular antioxidant systems, yet at higher doses it induces oxidative stress. Additionally, in vertebrates environmental exposures to toxic levels of selenium can cause paralysis and death. Here we show that selenium-induced oxidative stress leads to decreased cholinergic signaling and degeneration of cholinergic neurons required for movement and egg-laying in Caenorhabditis elegans. Exposure to high levels of selenium leads to proteolysis of a soluble muscle protein through mechanisms suppressible by two pharmacological agents, levamisole and aldicarb which enhance cholinergic signaling in muscle. In addition, animals with reduction-of-function mutations in genes encoding post-synaptic levamisole-sensitive acetylcholine receptor subunits or the vesicular acetylcholine transporter developed impaired forward movement faster during selenium-exposure than normal animals, again confirming that selenium reduces cholinergic signaling. Finally, the antioxidant reduced glutathione, inhibits selenium-induced reductions in egg-laying through a cellular protective mechanism dependent on the C. elegans glutaredoxin, GLRX-21. These studies provide evidence that the environmental toxicant selenium induces neurodegeneration of cholinergic neurons through depletion of glutathione, a mechanism linked to the neuropathology of Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease.
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PMID:Selenium induces cholinergic motor neuron degeneration in Caenorhabditis elegans. 2256 Sep 97

The intramolecular disulfide bond in hSOD1 [human SOD1 (Cu,Zn superoxide dismutase 1)] plays a key role in maintaining the protein's stability and quaternary structure. In mutant forms of SOD1 that cause familial ALS (amyotrophic lateral sclerosis), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalysed by CCS1 (copper chaperone for SOD1). Previous studies in yeast demonstrate that the yeast GSH/Grx (glutaredoxin) redox system promotes reduction of the hSOD1 disulfide in the absence of CCS1. In the present study, we probe further the interaction between hSOD1, GSH and Grxs to provide mechanistic insight into the redox kinetics and thermodynamics of the hSOD1 disulfide. We demonstrate that hGrx1 (human Grx1) uses a monothiol mechanism to reduce the hSOD1 disulfide, and the GSH/hGrx1 system reduces ALS mutant SOD1 at a faster rate than WT (wild-type) hSOD1. However, redox potential measurements demonstrate that the thermodynamic stability of the disulfide is not consistently lower in ALS mutants compared with WT hSOD1. Furthermore, the presence of metal cofactors does not influence the disulfide redox potential. Overall, these studies suggest that differences in the GSH/hGrx1 reaction rate with WT compared with ALS mutant hSOD1 and not the inherent thermodynamic stability of the hSOD1 disulfide bond may contribute to the greater pathogenicity of ALS mutant hSOD1.
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PMID:Redox properties of the disulfide bond of human Cu,Zn superoxide dismutase and the effects of human glutaredoxin 1. 2265 Oct 90

Modification of reactive cysteine residues plays an integral role in redox-regulated reactions. Oxidation of thiolate anions to sulphenic acid can result in disulphide bond formation, or overoxidation to sulphonic acid, representing reversible and irreversible endpoints of cysteine oxidation, respectively. The antioxidant systems of the cell, including the thioredoxin and glutaredoxin systems, aim to prevent these higher and irreversible oxidation states. This is important as these redox transitions have numerous roles in regulating the structure/function relationship of proteins. Proteins with redox-active switches as described for peroxiredoxin (Prx) and protein disulphide isomerase (PDI) can undergo dynamic structural rearrangement resulting in a gain of function. For Prx, transition from cysteine sulphenic acid to sulphinic acid is described as an adaptive response during increased cellular stress causing Prx to form higher molecular weight aggregates, switching its role from antioxidant to molecular chaperone. Evidence in support of PDI as a redox-regulated chaperone is also gaining impetus, where oxidation of the redox-active CXXC regions causes a structural change, exposing its hydrophobic region, facilitating polypeptide folding. In this review, we will focus on these two chaperones that are directly regulated through thiol-disulphide exchange and detail how these redox-induced switches allow for dual activity. Moreover, we will introduce a new role for a metabolic protein, the branched-chain aminotransferase, and discuss how it shares common mechanistic features with these well-documented chaperones. Together, the physiological importance of the redox regulation of these proteins under pathological conditions such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis will be discussed to illustrate the impact and importance of correct folding and chaperone-mediated activity.
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PMID:The redox switch that regulates molecular chaperones. 2635 57

Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys(57) and Cys(146), which have been linked to protein stability and folding via forming a disulfide bond, and Cys(6) and Cys(111) as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS.
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PMID:Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis. 2726 61

Cellular structural biology methods are needed to characterize biological processes at atomic resolution in the physiological environment of the cell. Toward this goal, solution in-cell NMR is a powerful approach because it provides structural and dynamic data on macromolecules inside living cells. Several approaches have been developed for in-cell NMR in cultured human cells, which are needed to study processes related to human diseases that rely on the delivery of exogenous macromolecules to the cells. Such strategies, however, may not be applicable to proteins that are sensitive to the external environment or prone to aggregate and can introduce artifacts during protein purification or delivery. As a complementary approach, direct protein expression for in-cell NMR in human cells was developed. This strategy is especially useful when studying processes like protein folding, maturation, and post-translational modification, starting right after protein synthesis. Compared with the protein expression techniques in mammalian cells commonly used in cellular biology, the low sensitivity of NMR requires higher protein levels. Among the cell lines used for high-yield protein expression, the HEK293T cell line was chosen, as it can be efficiently transfected with a cost-effective reagent. A vector originally designed for secreted proteins allows high-level cytosolic protein expression. For isotopic labeling, commercially available or homemade labeled media are employed. Uniform or amino acid type-selective labeling strategies are possible. Protein expression can be targeted to specific organelles (e.g., mitochondria), allowing for in organello NMR applications. A variant of the approach was developed that allows the sequential expression of two or more proteins, with only one selectively labeled. Protein expression in HEK293T cells was applied to recapitulate the maturation steps of intracellular superoxide dismutase 1 (SOD1) and to study the effect of mutations linked to familial amyotrophic lateral sclerosis (fALS) by in-cell NMR. Intracellular wild-type SOD1 spontaneously binds zinc, while it needs the copper chaperone for superoxide dismutase (CCS) for copper delivery and disulfide bond formation. Some fALS-linked mutations impair zinc binding and cause SOD1 to irreversibly unfold, likely forming the precursor of cytotoxic aggregates. The SOD-like domain of CCS acts as a molecular chaperone toward mutant SOD1, stabilizing its folding and allowing zinc binding and correct maturation. Changes in protein redox state distributions can also be investigated by in-cell NMR. Mitochondrial proteins require the redox-regulating partners glutaredoxin 1 (Grx1) and thioredoxin (Trx) to remain in the reduced, import-competent state in the cytosol, whereas SOD1 requires CCS for disulfide bond formation. In both cases, the proteins do not equilibrate with the cytosolic redox pool. Cysteine oxidation in response to oxidative stress can also be monitored. In the near future, in-cell NMR in human cells will likely benefit from technological advancements in NMR hardware, the development of bioreactor systems for increased sample lifetime, the application of paramagnetic NMR to obtain structural restraints, and advanced tools for genome engineering and should be increasingly integrated with advanced cellular imaging techniques.
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PMID:In-Cell NMR in Human Cells: Direct Protein Expression Allows Structural Studies of Protein Folding and Maturation. 2986 2