Intermolecular transmission of superoxide dismutase 1 misfolding in living cells

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and β-strand 3 of the SOD1 β-barrel (residues 24-36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein-protein interaction.     

Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to the misfolding of Cu/Zn superoxide dismutase (SOD1). ALS-related defects in SOD1 result in a gain of toxic function that coincides with aberrant oligomerization. The structural events triggering oligomerization have remained enigmatic, however, as is the case in other protein-misfolding diseases. Here, we target the critical conformational change that defines the earliest step toward aggregation. Using nuclear spin relaxation dispersion experiments, we identified a short-lived (0.4 ms) and weakly populated (0.7%) conformation of metal-depleted SOD1 that triggers aberrant oligomerization. This excited state emanates from the folded ground state and is suppressed by metal binding, but is present in both the disulfide-oxidized and disulfide-reduced forms of the protein. Our results pinpoint a perturbed region of the excited-state structure that forms intermolecular contacts in the earliest nonnative dimer/oligomer. The conformational transition that triggers oligomerization is a common feature of WT SOD1 and ALS-associated mutants that have widely different physicochemical properties. But compared with WT SOD1, the mutants have enhanced structural distortions in their excited states, and in some cases slightly higher excited-state populations and lower kinetic barriers, implying increased susceptibility to oligomerization. Our results provide a unified picture that highlights both (i) a common denominator among different SOD1 variants that may explain why diverse mutations cause the same disease, and (ii) a structural basis that may aid in understanding how different mutations affect disease propensity and progression.     

Atomic structures of wild-type and thermostable mutant recombinant human Cu,Zn superoxide dismutase.

Superoxide dismutase enzymes protect aerobic organisms from oxygen-mediated free-radical damage. Crystallographic structures of recombinant human Cu,Zn superoxide dismutase have been determined, refined, and analyzed at 2.5 A resolution for wild-type and a designed thermostable double-mutant enzyme (Cys-6----Ala, Cys-111----Ser). The 10 subunits (five dimers) in the crystallographic asymmetric unit form an unusual stable open lattice with 80-A-diameter channels. The 10 independently fit and refined subunits provide high accuracy, error analysis, and insights on loop conformations. There is a helix dipole interaction with the Zn site, and 14 residues form two or more structurally conserved side-chain to main-chain hydrogen bonds that appear critical to active-site architecture, loop conformation, and the increased stability resulting from the Cys-111----Ser mutation.     

Human Cu/Zn superoxide dismutase gene family: molecular structure and characterization of four Cu/Zn superoxide dismutase-related pseudogenes.

Four distinct human Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1)-related sequences were isolated from genomic DNA libraries. Genomic blots, heteroduplex analyses, and DNA sequencing showed that they are processed pseudogenes not residing on chromosome 21. Three of them originated from the 0.7-kilobase SOD1 mRNA, while the fourth was derived from the 0.9-kilobase mRNA species. Comparison between the coding sequences of the functional gene and two of the processed genes suggested that they integrated into the genome about 25 million years ago.     

Social interaction-mediated lifespan extension of Drosophila Cu/Zn superoxide dismutase mutants

Beneficial effects of social interaction on aging have been studied in humans and other species. We found that short-lived Drosophila mutants of the antioxidant enzyme Cu/Zn superoxide dismutase displayed a robust lifespan extension, with improved stress resistance and motor ability, upon cohousing with active flies of longer lifespan or younger age. Genetic, surgical, and environmental manipulations revealed motor and sensory components in behavioral interactions required for the lifespan extension induced by cohousing. Our results provide a definitive case of beneficial social interaction on lifespan and a useful entry point for analyzing the underlying molecular networks and physiological mechanisms.     

Dynamical roles of metal ions and the disulfide bond in Cu, Zn superoxide dismutase folding and aggregation

Misfolding and aggregation of Cu, Zn superoxide dismutase (SOD1) is implicated in neuronal death in amyotrophic lateral sclerosis. Each SOD1 monomer binds to 1 copper and 1 zinc ion and maintains its disulfide bond (Cys-57–Cys-146) in the reducing cytoplasm of cell. Mounting experimental evidence suggests that metal loss and/or disulfide reduction are important for initiating misfolding and aggregation of SOD1. To uncover the role of metals and the disulfide bond in the SOD1 folding, we systemically study the folding thermodynamics and structural dynamics of SOD1 monomer and dimer with and without metal binding and under disulfide-intact or disulfide-reduced environments in computational simulations. We use all-atom discrete molecular dynamics for sampling. Our simulation results provide dynamical evidence to the stabilizing role of metal ions in both dimer and monomer SOD1. The disulfide bond anchors a loop (Glu-49 to Asn-53) that contributes to the dimer interface. The reduction of the disulfide bond in SOD1 with metal ions depleted results in a flexible Glu-49–Asn-53 loop, which, in turn, disrupts dimer formation. Interestingly, the disulfide bond reduction does not affect the thermostability of monomer SOD1 as significantly as the metal ions do. We further study the structural dynamics of metal-free SOD1 monomers, the precursor for aggregation, in simulations and find inhomogeneous local unfolding of β-strands. The strands protected by the metal-binding and electrostatic loops are found to unfold first after metal loss, leading to a partially unfolded β-sheet prone to aggregation. Our simulation study sheds light on the critical role of metals and disulfide bond in SOD1 folding and aggregation.     

Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy

Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALS-mutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.     

Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40-100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.     

猪囊尾蚴铜/锌超氧化物歧化酶(Cu/ZnSOD)基因的克隆和表达

目的克隆猪囊尾蚴胞质含铜／锌的超氧化物歧化酶基因（Cu／ZnSOD），并比较其与其它寄生蠕虫相应基因结构的相似性。方法通过获取其它生物Cu／ZnSOD基因的保守区域，设计保守引物，用于扩增该寄生虫的Cu／ZnSOD基因。结果Cu／ZnSOD基因编码-15．6kDa的蛋白，该蛋白的推导序列中含有这类酶的活性和二级结构所需要的所有保守的氨基酸残基，并且与其它寄生虫的相应序列有高达70．6％的相似度。抑制剂研究试验表明所克隆的SOD属于Cu／ZnSOD，该蛋白在大肠杆菌中得以成功表达，表达产物具有SOD活性。免疫印迹试验发现猪囊尾蚴Cu／ZnSOD对人不仅具有抗原性，而且与其它几种蠕虫的Cu／ZnSOD有广泛的交叉免疫反应。结论克隆并表达出猪囊尾蚴Cu／ZnSOD，它与其它寄生吸虫和绦虫的Cu／ZnSOD在氨基酸水平具有很高的相似度，因此是理想的广谱抗寄生虫药物和疫苗的靶蛋白，值得进一步研究。     

Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells

Deposition of proteins of aberrant conformation is the hallmark of many neurodegenerative diseases. Misfolding of the normally globular mutant superoxide dismutase-1 (SOD1) is a central, early, but poorly understood event in the pathogenic cascade leading to familial forms of ALS. Here we report that aggregates composed of an ALS-causing SOD1 mutant penetrate inside cells by macropinocytosis and rapidly exit the macropinocytic compartment to nucleate aggregation of the cytosolic, otherwise soluble, mutant SOD1 protein. Once initiated, mutant SOD1 aggregation is self-perpetuating. Mutant SOD1 aggregates transfer from cell to cell with remarkable efficiency, a process that does not require contacts between cells but depends on the extracellular release of aggregates. This study reveals that SOD1 aggregates, propagate in a prion-like manner in neuronal cells and sheds light on the mechanisms underlying aggregate uptake and cell-to-cell transfer.     

A common mechanism of PLP/DM20 misfolding causes cysteine-mediated endoplasmic reticulum retention in oligodendrocytes and Pelizaeus-Merzbacher disease

A large number of mutations in the human PLP1 gene lead to abnormal myelination and oligodendrocyte death in Pelizaeus-Merzbacher disease (PMD). Here we show that a major subgroup of PMD mutations that map into the extracellular loop region of PLP/DM20 leads to the failure of oligodendrocytes to form the correct intramolecular disulfide bridges. This leads to abnormal protein cross-links and endoplasmic reticulum retention and activates the unfolded protein response. Importantly, surface expression of mutant PLP/DM20 can be restored and the unfolded protein response can be reverted by the removal of two cysteines. Thus, covalent protein cross-links emerge as a cause, rather than as a consequence, of endoplasmic reticulum retention.     

Cu, Zn superoxide dismutase deficiencyaccelerates the time course of an age-related markerin Drosophila melanogaster

In the oxidative stress hypothesis of aging therandom accumulation of oxidative damage over time ispostulated to cause aging. The pace at whichoxidative damage accrues determines the rate of aging,but it is less clear how the accumulation of randomdamage could cause the stereotypic pattern of aging. It has been proposed that oxidative damage induceschanges in gene expression, translating a random inputof damage into a patterned output. In support of thiswe show that in adult Drosophila melanogaster,with a deficiency in the anti-oxidant enzyme Cu, Znsuperoxide dismutase (Sod), an increase in oxidativestress, and a shortened life span, there isacceleration in the normal age-related temporalpattern of wingless gene expression. Theacceleration in the temporal pattern of winglessgene expression is proportional to the shortened lifespan suggesting that the shortened life span of Soddeficient animals is due, not to an abnormalpathological process, but to an increase in the rateof aging.     

Blood activity of Cu/Zn superoxide dismutase, glutathione peroxidase and catalase in Alzheimer's disease: a case-control study

Cu/Zn superoxide dismutase (Cu/Zn SOD), glutathione peroxidase (GPx) and catalase, which are the three main enzymes involved in cellular protection against damage due to oxygen-derived free radicals have been assayed in plasma and erythrocytes obtained from subjects with dementia of the Alzheimer type (DAT) and from controls. Blood samples were obtained from 25 patients with DAT and from age-matched subjects without diagnoses of neurological disease (non-DAT), as well as from younger individuals (reference group). Using appropriate statistical procedures, the three enzyme activities measured in blood of the elderly were decreased if compared to the younger reference group. Moreover, a significant increase in erythrocyte Cu/Zn SOD and catalase activities of DAT patients was observed compared to the non-DAT group. These results are discussed taking the free radical theory of aging into consideration.     

人铜锌超氧化物歧化酶原核表达载体的构建、表达及蛋白纯化

目的 构建人铜锌超氧化物歧化酶(hCu,Zn-SOD)原核表达载体并诱导其表达,纯化及鉴定目的 蛋白,为其临床应用奠定基础.方法 根据已报道的hCu,Zn-SOD基因序列,采用RT-PCR技术从外周血单个核细胞(PBMC)中获得SOD cDNA序列.将所得的PCR产物插入克隆载体pGEM-Teasy中,重组质粒经酶切,PCR及测序鉴定正确后,将目的 片段插入原核表达载体PET20 b( )中并转化大肠杆菌DE3,通过IPTG诱导表达出目的 蛋白,经镍固定金属亲和层析纯化.采用邻苯三酚自身氧化法测定SOD生物学活性.结果 序列分析表明SOD基因成熟肽编码区含有465 bp与GenBank(X02317)中已报道序列一致的SOD核苷酸.经IPTG诱导表达,SDS-PAGE电泳和免疫印迹分析显示表达的目的 蛋白分子质量约为19 kD并与商品化的人SOD单抗呈特异性反应.经Ni2 -NTA琼脂糖纯化获得SDS-PAGE电泳下单一条带.可溶蛋白酶活力达1 300 U/ml.结论 在大肠杆菌中获得了人SOD的高效表达,为研究其生物学功能和广泛应用奠定了基础.     

Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase

Several lines of evidence point to an abnormality in the response of Fanconi anemia cells to reactive oxygen species. To investigate the potential pathologic consequences of an in vivo alteration of redox state in mice lacking one of the Fanconi anemia genes, animals were generated having combined deficiencies of the cytosolic Cu/Zn superoxide dismutase (Sod1) and Fanconi anemia complementation group C (Fancc) genes. Interestingly, hepatocytes of Fancc(-/-)Sod1(-/-) mice exhibited a zonal pattern of microvesicular steatosis, possibly as a result of oxidative stress-induced injury to hepatocyte membranes. Consistent with this idea, freshly explanted Fancc(-/-)Sod1(-/-) hepatocytes demonstrated increased spontaneous production of superoxide in vitro. The second phenotypic feature of Fancc(-/-) Sod1(-/-) mice was that of bone marrow hypocellularity accompanied by significant decreases in peripheral blood erythrocyte and leukocyte numbers as compared with wild-type controls. Although flow cytometry analysis with monoclonal antibodies against cell surface antigens revealed normal numbers of primitive hematopoietic progenitor populations in Fancc(-/-)Sod1(-/-) marrow, lineage-positive progenitor numbers were significantly reduced in these mice. Furthermore, the in vitro clonogenic growth of Fancc(-/-)Sod1(-/-) erythroid, myeloid, and early B-lymphoid colonies in semisolid media was profoundly compromised. These results suggested that the altered redox state likely present in Fancc(-/-) Sod1(-/-) hematopoietic progenitors was responsible for an impairment of cell proliferation or survival. (Blood. 2001;98:1003-1011)     

Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP

OBJECTIVE: Previous studies from our group have shown a deficit in nitric oxide (NO) bioavailability and an excess production of the superoxide anion (O(2)(-)) in the stroke prone spontaneously hypertensive rat (SHRSP) compared to the normotensive Wistar Kyoto (WKY) strain. This present study has investigated whether adenoviral-mediated gene transfer of human eNOS or Cu/ZnSOD can alter the NO/O(2)(-) balance, thereby improving endothelial function. METHODS: A recombinant adenovirus, Ad/Hu/eNOS, containing the human eNOS cDNA fragment was generated by homologous recombination in 293 cells. Ad/Hu/eNOS or Ad/Cu/ZnSOD was delivered into SHRSP carotid arteries in vivo, using a titre of 2x10(9)-2x10(10) plaque forming units (pfu)/ml, and the effect on gene expression was observed 24 h later. RESULTS: Western blotting confirmed increased enzyme levels of eNOS and Cu/ZnSOD in the viral-infused vessels. Ex vivo, the pressor response to phenylephrine (PE) in the presence of L-NAME was increased in the eNOS-infused arteries relative to the contralateral controls, indicating restoration of basal NO availability to that observed in untreated control WKY rats. Infusion of the SOD virus produced a statistically insignificant increase in NO bioavailability. CONCLUSIONS: Our results support our previous findings obtained using a bovine eNOS recombinant adenovirus, that recombinant adenoviral gene transfer of human eNOS has a significant effect on NO bioavailability. In contrast, AdCu/ZnSOD gene transfer does not elicit an effect in our model. These results indicate that short-term overexpression of a recombinant eNOS, but not Cu/ZnSOD gene, in carotid arteries of the SHRSP is an effective means of locally increasing NO bioavailability to improve endothelial function.     

Neonatal mouse cardiac myocytes exhibit cardioprotection induced by hypoxic and pharmacologic preconditioning and by transgenic overexpression of human Cu/Zn superoxide dismutase

Although mouse models have been increasingly used for studies of cardiac pathophysiology, there is little information regarding cultured murine cardiac myocytes. Accordingly, we have developed a cell culture model of neonatal mouse cardiac myocytes by modifying a protocol used to prepare neonatal rat myocytes. The principal change is the substitution of cytosine arabinoside for bromodeoxyuridine to prevent fibroblast proliferation. Neonatal murine myocytes exhibited persistent spontaneous contraction and were viable for up to 14 days in culture. By flow cytometry 85% of the cells were cardiac myocytes. In sparse cultures (average cell density 259 cells/mm(2)), both hypoxic preconditioning (n=5) and phenylephrine pretreatment (n=8) produced significant protection of cardiac myocytes from cell death during a prolonged period of severe hypoxia (<0.5% O(2)for 18-20 h, both P<0.05). The phenylephrine effect was inhibited by the alpha(1)-adrenoceptor antagonist prazosin (n=4, P<0.05) and by an xi PKC peptide antagonist (xi V1-2) coupled to a TAT peptide (n=5, P<0. 05). Interestingly, the mixed alpha(1)- and beta -adrenoceptor agonist norepinephrine, which stimulates hypertrophy as measured by(14)[C]phenylalanine incorporation in neonatal rat cardiac myocytes, did not cause hypertrophy in mouse myocytes, suggesting that the signaling pathways for myocardial protection and hypertrophy are likely to be both divergent and species specific. In cardiac myocytes prepared from transgenic mice either homozygous or heterozygous for human Cu/Zn superoxide dismutase, there was protection from cell death (n=3) and restoration of(14)[C]phenyl- alanine uptake (n=4) during prolonged hypoxia (1% O(2)for 3 days, both P<0.05). We conclude that this cellular model, which is relatively simple to prepare, can be used for in-vitro examination of cardiac protection induced by preconditioning agents, various transgenes, and potentially by targeted gene deletions.     

Weakness of whole muscles in mice deficient in Cu, Zn superoxide dismutase is not explained by defects at the level of the contractile apparatus

Mice deficient in Cu,Zn superoxide dismutase (Sod1^−/− mice) demonstrate elevated oxidative stress associated with rapid age-related declines in muscle mass and force. The decline in mass for muscles of Sod1^−/− mice is explained by a loss of muscle fibers, but the mechanism underlying the weakness is not clear. We hypothesized that the reduced maximum isometric force (F_o) normalized by cross-sectional area (specific F_o) for whole muscles of Sod1^−/− compared with wild-type (WT) mice is due to decreased specific F_o of individual fibers. Force generation was measured for permeabilized fibers from muscles of Sod1^−/− and WT mice at 8 and 20 months of age. WT mice were also studied at 28 months to determine whether any deficits observed for fibers from Sod1^−/− mice were similar to those observed in old WT mice. No effects of genotype were observed for F_o or specific F_o at either 8 or 20 months, and no age-associated decrease in specific F_o was observed for fibers from Sod1^−/− mice, whereas specific F_o for fibers of WT mice decreased by 20 % by 28 months. Oxidative stress has also been associated with decreased maximum velocity of shortening (V_max), and we found a 10 % lower V_max for fibers from Sod1^−/−compared with WT mice at 20 months. We conclude that the low specific F_o of muscles of Sod1^−/− mice is not explained by damage to contractile proteins. Moreover, the properties of fibers of Sod1^−/− mice do not recapitulate those observed with aging in WT animals.     

浙贝母碱对肺癌A549/DDP细胞多药耐药的逆转作用观察及机制探讨

目的观察浙贝母碱对肺癌A549/DDP细胞多药耐药的逆转作用,并探讨其可能的机制。方法将对数生长期的细胞随机分成2组,对照组分别加入不同浓度的DDP,实验组在对照组基础上再加入400μg/mL的浙贝母碱;采用MTT法测算细胞增殖抑制率,计算两组细胞DDP的半数抑制浓度(IC50)及浙贝母碱的逆转倍数。将对数生长期的细胞分为3组,空白对照组不加任何药物,对照组加入DDP,实验组在对照组基础上再加入400μg/mL的浙贝母碱;药物作用48 h后,采用流式细胞术测算细胞凋亡率,用细胞免疫荧光法检测细胞中的人肺耐药蛋白(LRP)。结果实验组DDP的IC50为4.15μg/mL,对照组为15.46μg/mL,浙贝母碱的逆转耐药倍数为3.73。培养48 h后,空白对照组细胞凋亡率为2.56%±0.44%、LRP蛋白阳性细胞为(9.8±1.92)个/HP,对照组分别为为13.5%±1.42%、(9.2±1.9)个/HP,实验组为38.16%±2.25%、(5.8±1.3)个/HP;实验组与对照组、空白对照组比较,P均<0.05。结论浙贝母碱可逆转肺癌A549/DDP细胞株的多药耐药,其作用机制可能与其促进耐药细胞凋亡、下调LRP蛋白表达有关。