Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension

Olsson, Ingmar; Larsson, K; Palmgren, Ronnie; Bjellqvist, Bengt

doi:10.1002/1615-9861(200211)2:11<1630::aid-prot1630>3.0.co;2-n

Cited by 115 publications

(81 citation statements)

References 3 publications

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“…Two-dimensional Gel Electrophoresis (2DE)-The samples were dissolved in 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (v/v) immobilized pH gradient (IPG) buffer (Amersham Biosciences), and 12 l/ml DeStreak TM Reagent (Amersham Biosciences), which form stable disulfide bonds and prevent unspecific Cys residue oxidation during isoelectric focusing (IEF) (26). The samples were loaded by in-gel rehydration (1 h at 0 V, 270 Vhr at 30 V) on pH 4 -7 linear or pH 3-10 nonlinear 7-cm IPG strips (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Basso

Massignan

Giuseppina

et al. 2006

Journal of Biological Chemistry

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Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset. However, very little is known of the constituents and mechanism of formation of aggregates in ALS. We and others have shown that there is a progressive accumulation of detergent-insoluble mutant SOD1 in the spinal cord of G93A SOD1 mice. To investigate the mechanism of SOD1 aggregation, we characterized by proteome technologies SOD1 isoforms in a Triton X-100-insoluble fraction of spinal cord from G93A SOD1 mice at different stages of the disease. This showed that at symptomatic stages of the disease, part of the insoluble SOD1 is unambiguously mono-and oligoubiquitinated, in spinal cord and not in hippocampus, and that ubiquitin branches at Lys 48 , the major signal for proteasome degradation. At presymptomatic stages of the disease, only insoluble unmodified SOD1 is recovered. Partial ubiquitination of SOD1-rich inclusions was also confirmed by immunohistochemical and electron microscopy analysis of lumbar spinal cord sections from symptomatic G93A SOD1 mice. On the basis of these results, we propose that ubiquitination occurs only after SOD1 aggregation and that oligoubiquitination may underline alternative mechanisms in disease pathogenesis.

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Section: Methodsmentioning

confidence: 99%

Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Basso

Massignan

Giuseppina

et al. 2006

Journal of Biological Chemistry

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“…Samples containing 200 g of protein were diluted to a final volume of 450 l in a rehydration buffer consisting of 5 M urea (Merck), 2 M thiourea (Sigma), 2% CHAPS, 0.002% bromphenol blue, 0.8% IPG Buffer (pH 3-10) (Amersham Biosciences), and 1 tablet of CompleteMini TM protease inhibitor mixture (Roche Applied Science) per 1.5 ml. For reduction and alkylation of cysteines, a one-step method using dithiodiethanol was employed (14). Dithiodiethanol (Fluka, Germany) was added 20 min prior to starting the isoelectric focusing to a final concentration of 100 mM.…”

Section: Cell Culture and Generation Of Stable Transfectants-humanmentioning

confidence: 99%

Analysis of NOD2-mediated Proteome Response to Muramyl Dipeptide in HEK293 Cells

Weichart

Gobom

Klopfleisch

et al. 2006

Journal of Biological Chemistry

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WT that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2WT and disease-associated NOD2 SNP13 variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.NOD2 belongs to a growing family of regulatory nucleotide-binding oligomerization domain proteins with a central nucleotide-binding oligomerization domain and N-terminal caspase recruitment domains that are involved in programmed cell death and immune responses. The domain structure consists of two adjacent N-terminal caspase recruitment domains, a central nucleotide binding domain, and 10 C-terminal leucine-rich repeats (1). The leucine-rich repeats of NOD2 are homologous to those seen in R proteins and Toll-like receptors, which recognize pathogen-associated molecular patterns, thus enabling an innate cellular response to pathogens. Muramyl dipeptide (MurNAc-L-Ala-Diso-Gln, MDP-LD) 2 derived from peptidoglycan was recently identified as the ligand of NOD2 (2, 3). Upon MDP stimulation, NOD2 has been shown to activate the transcription factor nuclear factor B (NF-B) by interacting with the RIP-like interacting CLARP kinase (RICK/RIP2) (4).A frameshift mutation in the leucine-rich repeats of NOD2 (L1007fsinsC, SNP13), which leads to a partial truncation of the leucinerich repeats in the protein, has been associated with the development of Crohn disease, a human chronic relapsing-remitting inflammatory bowel disease (5-7). The truncation leads to a decreased MDP responsiveness and subsequent NF-B activation. Expression of NOD2 sensitizes intestinal epithelial cells to release the chemotactic cytokine IL-8 upon stimulation with bacterial cell wall components (8, 9). It has been shown recently that tumor necrosis factor-␣ up-regulates NOD2 in intestinal epithelial cells via an NF-B-dependent mechanism (8 -10). Nod2Ϫ/Ϫ mice are deficient in epithelial cryptdin expression and exhibit a higher susceptibility to oral Listeria infection (11). Thus, NOD2 has been implicated as a sentinel in maintaining the integrity of the intestinal barrier against luminal pathogens (12). However, the complex changes in protein expression subsequent to an activation of NF-B induced by NOD2 remain to be elucidated. We have addressed this task using a global proteomic approach. HEK293 cells were stably transfected with expression constructs encoding for the wild type form of NOD2 (NOD2 WT ) or the disease-associated NOD2 L1007fsinsC variant (NOD2 SNP13 ) stimulated with MDP-LD for 4 and 24 h, respectively, and protein extracts were analyzed by two-dimensional GE. Differentially regulated protein spots were detected by a semi-automated digital image analysis system, sampled from the gel, and the contained proteins subsequently were identified by MALDI-TOF pepti...

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“…PAGE of SDS-denatured proteins was performed as described by Laemmli (1970) (Figs 1 and 6b) and Schägger & von Jagow (1987); acrylamide/bis concentrations in the resolving gels were 15/0?1 and 10/0?3 %, respectively. Isoelectric focusing (IEF) was performed in immobilized pH gradients (13 cm pH 3-10 Immobiline DryStrips from Amersham Biosciences) in the presence of saturated urea, 50 mM DTT or hydroxyethyl disulfide (Olsson et al, 2002), and 1 % pH 3-10 ampholytes. Western blotting involved electrophoretic transfer to nitrocellulose or PVDF filters in the presence of 20 mM Tris/acetate (pH 8?3) and 20 % (v/v) methanol; antibody incubations and washes were performed in Trisbuffered saline (pH 7?5), 0?1 % gelatin and 0?1 % Tween 20.…”

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confidence: 99%

Yeast wall protein 1 of Candida albicans

et al. 2005

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Yeast wall protein 1 (Ywp1, also called Pga24) of Candida albicans is predicted to be a 533 aa polypeptide with an N-terminal secretion signal, a C-terminal glycosylphosphatidylinositol anchor signal and a central region rich in serine and threonine. In yeast cultures, Ywp1p appeared to be linked covalently to glucans of the wall matrix, but, as cultures approached stationary phase, Ywp1p accumulated in the medium and was extractable from cells with disulfide-reducing agents. An 11 kDa propeptide of Ywp1p was also present in these soluble fractions; it possessed the sole N-glycan of Ywp1p and served as a useful marker for Ywp1p. DNA vaccines encoding all or part of Ywp1p generated analytically useful antisera in mice, but did not increase survival times for disseminated candidiasis. Replacement of the coding sequence of YWP1 with the fluorescent reporter GFP revealed that expression of YWP1 is greatest during yeast exponential-phase growth, but downregulated in stationary phase and upon filamentation. Expression was upregulated when the extracellular phosphate concentration was low. Disruption by homologous recombination of both YWP1 alleles resulted in no obvious change in growth, morphology or virulence, but the Ywp1p-deficient blastoconidia exhibited increased adhesiveness and biofilm formation, suggesting that Ywp1p may promote dispersal of yeast forms of C. albicans.

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Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension

Cited by 115 publications

References 3 publications

Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Analysis of NOD2-mediated Proteome Response to Muramyl Dipeptide in HEK293 Cells

Yeast wall protein 1 of Candida albicans

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