Abstract:The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis. The chemical mechanism underlying this effect is unclear, hydrophobic/steric effects electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 has not been addressed. Here, we acylated a fraction of lysine residues in SOD1 with groups of variable hydroph… Show more
“…Transmission electron microscopy (TEM) revealed that G93A ThT binding was associated with fibril formation, whereas G93A-S32 TEM structures were sparsely distributed amorphous aggregates (Fig. 1C ), ruling out the generation of ThT negative SOD1 fibrils as has been reported 24 . Figure 1 S32 substitution mitigates aggregate formation of purified SOD1 mutants and lowers their stability.…”
Over 160 mutations in superoxide dismutase 1 (SOD1) are associated with familial amyotrophic lateral sclerosis (fALS), where the main pathological feature is deposition of SOD1 into proteinaceous cytoplasmic inclusions. We previously showed that the tryptophan residue at position 32 (W32) mediates the prion-like propagation of SOD1 misfolding in cells, and that a W32S substitution blocks this phenomenon. Here, we used in vitro protein assays to demonstrate that a W32S substitution in SOD1-fALS mutants significantly diminishes their propensity to aggregate whilst paradoxically decreasing protein stability. We also show SOD1-W32S to be resistant to seeded aggregation, despite its high abundance of unfolded protein. A cell-based aggregation assay demonstrates that W32S substitution significantly mitigates inclusion formation. Furthermore, this assay reveals that W32 in SOD1 is necessary for the formation of a competent seed for aggregation under these experimental conditions. Following the observed importance of W32 for aggregation, we established that treatment of living cells with the W32-interacting 5-Fluorouridine (5-FUrd), and its FDA approved analogue 5-Fluorouracil (5-FU), substantially attenuate inclusion formation similarly to W32S substitution. Altogether, we highlight W32 as a significant contributor to SOD1 aggregation, and propose that 5-FUrd and 5-FU present promising lead drug candidates for the treatment of SOD1-associated ALS.
“…Transmission electron microscopy (TEM) revealed that G93A ThT binding was associated with fibril formation, whereas G93A-S32 TEM structures were sparsely distributed amorphous aggregates (Fig. 1C ), ruling out the generation of ThT negative SOD1 fibrils as has been reported 24 . Figure 1 S32 substitution mitigates aggregate formation of purified SOD1 mutants and lowers their stability.…”
Over 160 mutations in superoxide dismutase 1 (SOD1) are associated with familial amyotrophic lateral sclerosis (fALS), where the main pathological feature is deposition of SOD1 into proteinaceous cytoplasmic inclusions. We previously showed that the tryptophan residue at position 32 (W32) mediates the prion-like propagation of SOD1 misfolding in cells, and that a W32S substitution blocks this phenomenon. Here, we used in vitro protein assays to demonstrate that a W32S substitution in SOD1-fALS mutants significantly diminishes their propensity to aggregate whilst paradoxically decreasing protein stability. We also show SOD1-W32S to be resistant to seeded aggregation, despite its high abundance of unfolded protein. A cell-based aggregation assay demonstrates that W32S substitution significantly mitigates inclusion formation. Furthermore, this assay reveals that W32 in SOD1 is necessary for the formation of a competent seed for aggregation under these experimental conditions. Following the observed importance of W32 for aggregation, we established that treatment of living cells with the W32-interacting 5-Fluorouridine (5-FUrd), and its FDA approved analogue 5-Fluorouracil (5-FU), substantially attenuate inclusion formation similarly to W32S substitution. Altogether, we highlight W32 as a significant contributor to SOD1 aggregation, and propose that 5-FUrd and 5-FU present promising lead drug candidates for the treatment of SOD1-associated ALS.
“…The agent employed for acetylation of SOD1 in vitro as well as the site(s) of acetylation influenced the type of aggregates formed [152]. SUMOylation at K75 increased aggregation of mutant (and wild-type) SOD1 [153,154] (Table 6 and Figure 6).…”
Section: Sod1 Ptms and Propensity For Aggregationmentioning
The accumulation of abnormal protein aggregates represents a universal hallmark of neurodegenerative diseases (NDDs). Post-translational modifications (PTMs) regulate protein structure and function. Dysregulated PTMs may influence the propensity for protein aggregation in NDD-proteinopathies. To investigate this, we systematically reviewed the literature to evaluate effects of PTMs on aggregation propensity for major proteins linked to the pathogenesis and/or progression of NDDs. A search of PubMed, MEDLINE, EMBASE, and Web of Science Core Collection was conducted to retrieve studies that investigated an association between PTMs and protein aggregation in seven NDDs: Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), spinocerebellar ataxias, transmissible spongiform encephalopathy, and multiple sclerosis. Together, 1222 studies were identified, of which 69 met eligibility criteria. We identified that the following PTMs, in isolation or combination, potentially act as modulators of proteinopathy in NDDs: isoaspartate formation in Aβ, phosphorylation of Aβ or tau in AD; acetylation, 4-hydroxy-2-neonal modification, O-GlcNAcylation or phosphorylation of α-synuclein in PD; acetylation or phosphorylation of TAR DNA-binding protein-43 in ALS, and SUMOylation of superoxide dismutase-1 in ALS; and phosphorylation of huntingtin in HD. The potential pharmacological manipulation of these aggregation-modulating PTMs represents an as-yet untapped source of therapy to treat NDDs.
“…Recently, we have also con rmed the declined levels of Sirt3 in the brain tissues of several scrapie infected mice and in SMB-S15 cells, leading to increases of acetylating forms of SOD2 and ATP5β that subsequently induce increase of intracellular ROS and reduction of ATP [20]. Aberrant alterations of acetylation modi cations for other proteins have been also found in the brain tissues or the models in vitro of several neurodegenerative diseases, e.g., tau, microtubules, SOD1 [21][22][23]. However, the global change of protein acetylation in the brain tissues of prion diseases remains unsettled.…”
BackgroundAcetylation is a reversible post-translational modification in eukaryotic and prokaryotic cells, actively participating in the regulation for biological functions and in the pathogenesis of diseases.MethodsThe acetylated proteins from the cortex regions of scrapie agents 139A- and ME7-infected mice collected at mid-early (80 days post-infection, dpi), mid-late (120 dpi) and terminal stage (180 dpi) were extracted. The global profiles of the brain acetylated proteins were assayed with proteomic mass spectrometry. The acetylated peptides whose levels were >1.5-fold higher or lower than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs).ResultsA total of 1,485 acetylated peptides were identified. 118, 42 and 51 DEAPs were in the brains of 139A-80 dpi, 120 dpi and 180 dpi, while 390, 227 and 75 DEAPs were in those of ME7-80 dpi, 120 dpi and 180 dpi, respectively. Overwhelming majority of the DEAPs in mid-early stage was down-regulated, while more portions of DEAPs in mid-late and late stage were up-regulated. Approximately 22.1% (328/1485) acetylated peptides were mitochondrial associated, which were mapped to 74 different proteins. Among them, 44 (59.5%) proteins showed differentially expressed at least at one tested time-point. KEEG pathways analysis identified 39, 13, 10 and 55, 25, 18 pathways in the samples of 80, 120 and 180 dpi of 139A- and ME7-infected mice as significantly changed (P<0.05), respectively. Six pathways were commonly involved in all tested samples, including carbon metabolism, metabolic pathways, biosynthesis of amino acids, glycolysis/gluconeogenesis, pyruvate metabolism and citrate cycle (TCA cycle). Moreover, dozens of steps in TAC cycle were affected via down-regulated acetylation for the relevant enzymes in the mid-early stage, while many steps were affected in the mid-late stage via up-regulated acetylation. In the late stage, the affected steps focused on up-regulated acetylation for succinate dehydrogenase, fumarate hydratase and malate dehydrogenase.ConclusionCollectively, Our data here illustrated a picture of global acetylation for brain proteins during prion infection, showing remarkably inhibiting acetylation in the early stage and relatively enhanced acetylation in the late stage.
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