Function of the Greek key connection analysed using circular permutants of superoxide dismutase

Boissinot, Maurice; Karnas, S; Lepock, James R.; Cabelli, Diane E.; Tainer, John A.; Getzoff, Elizabeth D.; Hallewell, Robert A.

doi:10.1093/emboj/16.9.2171

Cited by 34 publications

(25 citation statements)

References 48 publications

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“…Fully metallated SOD1 was delipidated using hydroxyalkoxypropyl dextran type III (Sigma-Aldrich) as described previously (19) before de-metallation. Metal-deficient apo-enzymes were prepared as described previously (23), and loss of enzyme activity was confirmed after de-metallation. The metal content of purified enzymes was estimated as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

Unsaturated Fatty Acids Induce Cytotoxic Aggregate Formation of Amyotrophic Lateral Sclerosis-linked Superoxide Dismutase 1 Mutants

Kim¹,

Nakatomi

Akagi

et al. 2005

Journal of Biological Chemistry

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Formation of misfolded protein aggregates is a remarkable hallmark of various neurodegenerative diseases including Alzheimer disease, Parkinson disease, Huntington disease, prion encephalopathies, and amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) immunoreactive inclusions have been found in the spinal cord of ALS animal models and patients, implicating the close involvement of SOD1 aggregates in ALS pathogenesis. Here we examined the molecular mechanism of aggregate formation of ALS-related SOD1 mutants in vitro. We found that long-chain unsaturated fatty acids (FAs) promoted aggregate formation of SOD1 mutants in both dose-and time-dependent manners. Metal-deficient SOD1s, wild-type, and mutants were highly oligomerized compared with holo-SOD1s by incubation in the presence of unsaturated FAs. Oligomerization of SOD1 is closely associated with its structural instability. Heat-treated holo-SOD1 mutants were readily oligomerized by the addition of unsaturated FAs, whereas wild-type SOD1 was not. The monounsaturated FA, oleic acid, directly bound to SOD1 and was characterized by a solid-phase FA binding assay using oleate-Sepharose. The FA binding characteristics were closely correlated with the oligomerization propensity of SOD1 proteins, which indicates that FA binding may change SOD1 conformation in a way that favors the formation of aggregates. High molecular mass aggregates of SOD1 induced by FAs have a granular morphology and show significant cytotoxicity. These findings suggest that SOD1 mutants gain FA binding abilities based on their structural instability and form cytotoxic granular aggregates. Amyotrophic lateral sclerosis (ALS)1 is a progressive and fatal neurodegenerative disorder that mainly affects motor neurons in the brain stem and spinal cord. Approximately 10% of ALS patients are familial cases, with autosomal dominant inheritance. More than 90 different mutations in the gene coding for superoxide dismutase 1 (SOD1) have been identified in about 20% of familial ALS (FALS) families (1, 2). Although the molecular mechanisms of selective motor neuron degeneration by SOD1 mutants in FALS remain largely unknown, common pathological features of conformational diseases, as evidenced by SOD1 immunoreactive inclusions, are found in the spinal cord of ALS patients and in the SOD1 mutant FALS mouse model (3-8). The characteristics of FALS resemble those of many other neurodegenerative diseases in which a causative protein undergoes a conformational rearrangement, which endows it with a tendency to aggregate and form deposits within affected tissues.SOD1 is a 32-kDa homodimeric enzyme that decreases the intracellular concentration of superoxide radicals by catalyzing their dismutation to O 2 and H 2 O 2 . ALS-linked mutations of SOD1 are distributed throughout the primary and tertiary structures, and most mutations appear unrelated to the dismutase activity. Many biochemical and biophysical studies have reported that SOD1 mutants are structurally unstable compared with wild-type forms (...

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Section: Methodsmentioning

confidence: 99%

Unsaturated Fatty Acids Induce Cytotoxic Aggregate Formation of Amyotrophic Lateral Sclerosis-linked Superoxide Dismutase 1 Mutants

Kim¹,

Nakatomi

Akagi

et al. 2005

Journal of Biological Chemistry

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“…The reaction was allowed to proceed at Ϫ5-0°C for 1 h. After cleavage, the peptides were dissolved in 50% acetonitrile, 0.1% trifluoroacetic acid and lyophilized. The crude, reduced peptides were purified using preparative reverse-phase HPLC (RP-HPLC) 1 on a Vydac C18 column. Gradients of 0.1% aqueous trifluoroacetic acid and 90% acetonitrile, 0.09% trifluoroacetic acid were employed with a flow rate of 8 ml/min, and the eluant was monitored at 230 nm.…”

Section: Methodsmentioning

confidence: 99%

“…It may be thought of as joining the ends of a protein and then opening the chain elsewhere. These experiments have shown that it is possible in some cases to permute the termini of a protein and still allow folding into the native conformation (1,2), thus providing valuable information on protein folding. In practice the conceptual "circular proteins" intrinsic to the design of circular permutants are never actually synthesized but are theoretical intermediates.…”

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confidence: 99%

Acyclic Permutants of Naturally Occurring Cyclic Proteins

Daly

Craik

2000

Journal of Biological Chemistry

104

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Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple-stranded ␤-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the ␤-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.

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“…Alkylated and separated EC-SOD monomers were sequentially dialyzed against 50 mM acetate͞10 mM EDTA, pH 3.8; 50 mM acetate͞100 mM NaCl, pH 3.8; 100 mM acetate, pH 3.8; 100 mM acetate, pH 5.5; 100 mM acetate͞50 M CuSO 4 ͞20 M ZnSO 4 , pH 5.5; and 10 mM Tris-HCl, pH 7.4 as described (20,21).…”

mentioning

confidence: 99%

The dual nature of human extracellular superoxide dismutase: One sequence and two structures

Petersen

Oury

Valnickova

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Human extracellular superoxide dismutase (EC-SOD; EC 1.15.1.1) is a scavenger of superoxide anions in the extracellular space. The amino acid sequence is homologous to the intracellular counterpart, Cu͞Zn superoxide dismutase (Cu͞Zn-SOD), apart from N-and C-terminal extensions. Cu͞Zn-SOD is a homodimer containing four cysteine residues within each subunit, and EC-SOD is a tetramer composed of two disulfide-bonded dimers in which each subunit contains six cysteines. The amino acid sequences of all EC-SOD subunits are identical. It is known that Cys-219 is involved in an interchain disulfide. To account for the remaining five cysteine residues we purified human EC-SOD and determined the disulfide bridge pattern. The results show that human EC-SOD exists in two forms, each with a unique disulfide bridge pattern. One form (active EC-SOD) is enzymatically active and contains a disulfide bridge pattern similar to Cu͞Zn-SOD. The other form (inactive EC-SOD) has a different disulfide bridge pattern and is enzymatically inactive. The EC-SOD polypeptide chain apparently folds in two different ways, most likely resulting in different three-dimensional structures. Our study shows that one gene may produce proteins with different disulfide bridge arrangements and, thus, by definition, different primary structures. This observation adds another dimension to the functional annotation of the proteome. T wo isoforms of Cu͞Zn-containing superoxide dismutase (SOD) enzymes exist in mammals (1, 2). Cu͞Zn-SOD is found in the intracellular space, and extracellular SOD (EC-SOD) predominantly is found in the extracellular matrix of most tissues (3). Both enzymes dismutate the superoxide anion into hydrogen peroxide and oxygen with diffusion-limited rate constants (Ͼ10 9 M Ϫ1 sec Ϫ1 ), and both are inhibited by cyanide and azide (4, 5). Human Cu͞Zn-SOD is a homodimer with a molecular mass of 32 kDa, and human EC-SOD is a tetramer of Ϸ135 kDa (4). The subunit of each isoform contains one Cu(II) and one Zn(II) atom. The central region of EC-SOD (His-96 to ) is homologous to human Cu͞Zn-SOD and contains all of the ligands essential for the coordination of the active site Cu(II) and Zn(II) ions (6, 7). The N-terminal region of EC-SOD is important for the formation of tetramers (8-10), and the C-terminal region ) encompasses a heparinbinding region, which is responsible for the immobilization of EC-SOD in the extracellular matrix (11, 12). The heparinbinding region of EC-SOD can be removed by an intracellular proteolytic event before secretion (13,14). Consequently, EC-SOD tetramers with no (type A), intermediate (type B), or high (type C) affinity for the extracellular matrix are produced (11,12). These tetramers are composed of cleaved polypeptides (type A), intact polypeptides (type C), or a mixture (type B). In addition, the C-terminal region supports the formation of disulfide-linked dimers via 16).The x-ray structure of Cu͞Zn-SOD shows that the active-site channel is maintained by an intrapolypeptide disulfide bond between two highly co...

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Function of the Greek key connection analysed using circular permutants of superoxide dismutase

Cited by 34 publications

References 48 publications

Unsaturated Fatty Acids Induce Cytotoxic Aggregate Formation of Amyotrophic Lateral Sclerosis-linked Superoxide Dismutase 1 Mutants

Unsaturated Fatty Acids Induce Cytotoxic Aggregate Formation of Amyotrophic Lateral Sclerosis-linked Superoxide Dismutase 1 Mutants

Acyclic Permutants of Naturally Occurring Cyclic Proteins

The dual nature of human extracellular superoxide dismutase: One sequence and two structures

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