scholarly journals A Novel SOD1 Intermediate Oligomer, Role of Free Thiols and Disulfide Exchange

2021 ◽  
Vol 14 ◽  
Author(s):  
Bon-Kyung Koo ◽  
William Munroe ◽  
Edith B. Gralla ◽  
Joan Selverstone Valentine ◽  
Julian P. Whitelegge
Keyword(s):  
Disulfide Bond ◽  
Metal Binding ◽  
Amyloid Fibrils ◽  
Wild Type ◽  
Disulfide Exchange ◽  
Metal Binding Sites ◽  
Disulfide Reduction ◽  
Intramolecular Disulfide Bond ◽  
Mixed Disulfides

Wild-type human SOD1 forms a highly conserved intra-molecular disulfide bond between C57-C146, and in its native state is greatly stabilized by binding one copper and one zinc atom per monomer rendering the protein dimeric. Loss of copper extinguishes dismutase activity and destabilizes the protein, increasing accessibility of the disulfide with monomerization accompanying disulfide reduction. A further pair of free thiols exist at C6 and C111 distant from metal binding sites, raising the question of their function. Here we investigate their role in misfolding of SOD1 along a pathway that leads to formation of amyloid fibrils. We present the seeding reaction of a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) to exclude variables caused by these free cysteines. Completely reduced fibril seeds decreasing the kinetic barrier to cleave the highly conserved intramolecular disulfide bond, and accelerating SOD1 reduction and initiation of fibrillation. Presence or absence of the pair of free thiols affects kinetics of fibrillation. Previously, we showed full maturation with both Cu and Zn prevents this behavior while lack of Cu renders sensitivity to fibrillation, with presence of the native disulfide bond modulating this propensity much more strongly than presence of Zn or dimerization. Here we further investigate the role of reduction of the native C57-C146 disulfide bond in fibrillation of wild-type hSOD1, firstly through removal of free thiols by paired mutations C6A, C111S (AS-SOD1), and secondly in seeded fibrillation reactions modulated by reductant tris (2-carboxyethyl) phosphine (TCEP). Fibrillation of AS-SOD1 was dependent upon disulfide reduction and showed classic lag and exponential growth phases compared with wild-type hSOD1 whose fibrillation trajectories were typically somewhat perturbed. Electron microscopy showed that AS-SOD1 formed classic fibrils while wild-type fibrillation reactions showed the presence of smaller “sausage-like” oligomers in addition to fibrils, highlighting the potential for mixed disulfides involving C6/C111 to disrupt efficient fibrillation. Seeding by addition of sonicated fibrils lowered the TCEP concentration needed for fibrillation in both wild-type and AS-SOD1 providing evidence for template-driven structural disturbance that elevated susceptibility to reduction and thus propensity to fibrillate.

scholarly journals Role of the Intramolecular Disulfide Bond in FlgI, the Flagellar P-Ring Component of Escherichia coli

2006 ◽  
Vol 188 (12) ◽  
pp. 4190-4197 ◽  
Author(s):  
Yohei Hizukuri ◽  
Toshiharu Yakushi ◽  
Ikuro Kawagishi ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Self Assembly ◽  
Bacterial Species ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Wild Type ◽  
Ring Assembly ◽  
Intramolecular Disulfide Bond

ABSTRACT The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli, because the P ring was not assembled in a dsbB strain that was defective for disulfide bond formation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved in other bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cys-eliminated FlgI derivative formed a P ring that complemented the flagellation defect of our ΔflgI strain when it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation than wild-type FlgI. Overproduction of FlgI suppressed the motility defect of ΔdsbB cells. Additionally, the low level of FlgI observed in the ΔdsbB strain increased in the presence of l-cystine, an oxidative agent. We propose that intramolecular disulfide bond formation facilitates the rapid folding of the FlgI monomer to protect against degradation in the periplasmic space, thereby allowing its efficient self-assembly into the P ring.

scholarly journals Catalysis of cataract-associated human γD crystallin aggregation via dynamic disulfide exchange

2018 ◽  
Author(s):  
Eugene Serebryany ◽  
Shuhuai Yu ◽  
Sunia A. Trauger ◽  
Bogdan Budnik ◽  
Eugene I. Shakhnovich
Keyword(s):  
Light Scattering ◽  
Disulfide Bond ◽  
Eye Lens ◽  
Lens Protein ◽  
Biochemical Mechanism ◽  
Wild Type ◽  
Disulfide Exchange ◽  
Mechanism Of Catalysis ◽  
Eye Lens Protein

AbstractSeveral mutations in human γD-crystallin (HγD), a long-lived eye lens protein, cause misfolding and aggregation, leading to cataract. Surprisingly, wild-type HγD catalyzes aggregation of its cataract related W42Q variant while itself remaining soluble – the inverse of the classical prion-like scenario whereby misfolded polypeptides catalyze aggregation of natively folded ones. The search for a biochemical mechanism of catalysis of W42Q aggregation by WT has revealed that WT HγD can transfer a disulfide bond to the W42Q variant. The transferred disulfide kinetically traps an aggregation-prone intermediate made accessible by the W42Q mutation, facilitating light-scattering aggregation of the W42Q variant. The aggregating variant thus becomes a disulfide sink, removing the disulfides from solution. Such redox “hot potato” competitions among wild-type and mutant or modified polypeptides may be relevant for many long-lived proteins that function in oxidizing environments. In these cases aggregation may be forestalled by inhibiting disulfide flow toward damaged polypeptides.

scholarly journals The crystal structure of wild-type human brain neuroglobin reveals flexibility of the disulfide bond that regulates oxygen affinity

2014 ◽  
Vol 70 (4) ◽  
pp. 1005-1014 ◽  
Author(s):  
Beatriz G. Guimarães ◽  
Djemel Hamdane ◽  
Christophe Lechauve ◽  
Michael C. Marden ◽  
Béatrice Golinelli-Pimpaneau
Keyword(s):  
Disulfide Bond ◽  
Conformational Transition ◽  
Bond Formation ◽  
Oxygen Affinity ◽  
Disulfide Bridge ◽  
Wild Type ◽  
X Ray ◽  
Intramolecular Disulfide Bond ◽  
Internal Cavities ◽  
High Oxygen Affinity

Neuroglobin plays an important function in the supply of oxygen in nervous tissues. In human neuroglobin, a cysteine at position 46 in the loop connecting the C and D helices of the globin fold is presumed to form an intramolecular disulfide bond with Cys55. Rupture of this disulfide bridge stabilizes bi-histidyl haem hexacoordination, causing an overall decrease in the affinity for oxygen. Here, the first X-ray structure of wild-type human neuroglobin is reported at 1.74 Å resolution. This structure provides a direct observation of two distinct conformations of the CD region containing the intramolecular disulfide link and highlights internal cavities that could be involved in ligand migration and/or are necessary to enable the conformational transition between the low and high oxygen-affinity states following S—S bond formation.

Role of Ecklonia radiata (C. Ag.) J. Agardh in determining trace metal availability in coastal waters. I. Total trace metals

1987 ◽  
Vol 38 (3) ◽  
pp. 307 ◽  
Author(s):  
HW Higgins ◽  
DJ Mackey
Keyword(s):  
Trace Metals ◽  
Metal Binding ◽  
Late Summer ◽  
Dry Weight ◽  
Metal Availability ◽  
Metal Binding Sites ◽  
Accumulation Of Metals ◽  
Concentration Factors

No seasonal variations were found in the concentrations of Zn, Cd, Cu, K, Ca, Mg and Na in the kelp E. radiata collected from the marine-dominated Port Hacking estuary on the east coast of Australia. Concentrations of Fe and Mn were about 60% higher in late summer. The relative distributions of all metals between different kelp tissues, however, showed no seasonal variation. Concentration factors (dry weight basis) for trace metals ranged from 2600 for Cu to 68 000 for Fe. With high biomasses common in macroalgal ecosystems, a large proportion of the non-sediment- bound trace metals can be associated with the macroalgae, which therefore act as substantial buffers for these elements. Metal concentration factors (Y, wet wt basis) were related to oceanic residence times (τ) by the equation log Y = -0.69 logτ + 5.4. The distribution of the aikaii and akaline earth metais (K, Ca, Mg and Na) was relativeiy uniform throughout the various kelp tissues. However, concentrations of Fe, Mn, Zn and Cd were significantly higher in the older extremities (holdfast and eroding tip) than in the meristematic region. Although the holdfast also had higher levels of Cu than the meristem, levels were lower in the eroding tip. The results suggest either a slow net intracellular accumulation of metals with time or an increase in potential metal- binding sites as the extremities senesce. Translocation and elimination of surplus metals through the eroding tip or holdfast was thought not to be important in E. radiata as metal concentrations did not differ between live and dead haptera of the holdfast. Likewise, storage of metals in either the holdfast or eroding tip was considered unlikely because of the constant relative tissue distribution throughout the year and lack of metal mobilization during periods of growth. Pretreatment of kelp tissue with an EDTA wash released about 90% of the total Zn and Cd, 25% of the Cu and 7% of the Fe, suggesting that a large proportion of the total kelp Zn and Cd is associated with the apparent free space (AFS). With rapid exchange between seawater and the AFS, E. radiata is therefore not generally useful as a sentinel accumulator species in pollution studies for assessing long term integrated changes of metals in the water column.

Metallothionein is a crucial protective factor againstHelicobacter pylori-induced gastric erosive lesions in a mouse model

2008 ◽  
Vol 294 (4) ◽  
pp. G877-G884 ◽  
Author(s):  
Masaharu Mita ◽  
Masahiko Satoh ◽  
Akinori Shimada ◽  
Mina Okajima ◽  
Sadahiro Azuma ◽  
...  
Keyword(s):  
Metal Binding ◽  
Protective Factor ◽  
Histopathological Examination ◽  
Inflammatory Cells ◽  
Gastric Injury ◽  
Wild Type ◽  
Inflammatory Protein ◽  
Ros Scavenger ◽  
H Pylori

Infection with the gastric pathogen Helicobacter pylori ( H. pylori) causes chronic gastritis, peptic ulcer, and gastric adenocarcinoma. These diseases are associated with production of reactive oxygen species (ROS) from infiltrated macrophages and neutrophiles in inflammatory sites. Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that can act not only as a metal-binding protein, but also as a ROS scavenger. In the present study, we examined the role of MT in the protection against H. pylori-induced gastric injury using MT-null mice. Female MT-null and wild-type mice were challenged with H. pylori SS1 strain, and then histological changes were evaluated with the updated Sydney grading system at 17 and 21 wk after challenge. Although the colonization efficiency of H. pylori was essentially the same for MT-null and wild-type mice, the scores of activity of inflammatory cells were significantly higher in MT-null mice than in wild-type mice at 17 wk after challenge. Histopathological examination revealed erosive lesions accompanied by infiltration of inflammatory cells in the infected MT-null mice but not in wild-type mice. Furthermore, activation of NF-κB and expression of NF-κB-mediated chemokines such as macrophage inflammatory protein-1α and monocytes chemoattractant protein-1 in gastric cells were markedly higher in MT-null mice than in wild-type mice. These results suggest that MT in the gastric mucosa might play an important role in the protection against H. pylori-induced gastric ulceration.

Asymmetric cell division and differentiation; fern spore germination as a model. II. Ultrastructural studies

1985 ◽  
Vol 86 ◽  
pp. 227-230
Author(s):  
Alix R. Bassel
Keyword(s):  
Cell Division ◽  
Metal Binding ◽  
Asymmetric Cell Division ◽  
Red Light ◽  
Nuclear Migration ◽  
Silver Stain ◽  
Metal Binding Sites ◽  
Ultrastructural Studies ◽  
Fern Spore Germination

SynopsisThe germination of Onoclea spores is a model system with many advantages for the study of asymmetric cell division and cellular differentiation. Our results suggest that both microtubules and a lipophilic site are important in the nuclear migration to one end of the spore prior to asymmetric cell division. A metalbinding region containing pore-like structures in the proximal face of the spore coat may be a source of the inherent polarity of the spore. The pattern of endogenous metal binding during germination has been characterised using a sulphide-silver stain. Metal-binding sites are described in a differentiating system in which polarity is imposed externally using polarised red light. The possibility of a role of ion gradients in determining the direction of nuclear migration is discussed.

scholarly journals Disulfide Bond Formation in Secreton Component PulK Provides a Possible Explanation for the Role of DsbA in Pullulanase Secretion

2001 ◽  
Vol 183 (4) ◽  
pp. 1312-1319 ◽  
Author(s):  
Anthony P. Pugsley ◽  
Nicolas Bayan ◽  
Nathalie Sauvonnet
Keyword(s):  
Disulfide Bond ◽  
Klebsiella Oxytoca ◽  
Bond Formation ◽  
Type Ii Secretion ◽  
Disulfide Bond Formation ◽  
Gene Encoding ◽  
Intramolecular Disulfide Bond ◽  
Disulfide Oxidoreductase ◽  
Considerable Excess

ABSTRACT When expressed in Escherichia coli, the 15Klebsiella oxytoca pul genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these pul genes, efficient pullulanase secretion also requires the host dsbA gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by adsbA mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a dsbA mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.

scholarly journals The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

2002 ◽  
Vol 70 (9) ◽  
pp. 4968-4976 ◽  
Author(s):  
Susanna Ricci ◽  
Robert Janulczyk ◽  
Lars Björck
Keyword(s):  
Oxidative Stress ◽  
Streptococcus Pyogenes ◽  
Stress Responses ◽  
Metal Binding ◽  
Iron Homeostasis ◽  
Bacterial Species ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Allelic Replacement

ABSTRACT Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of Streptococcus pyogenes. To determine the role of PerR in S. pyogenes, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of 55Fe incorporation from the culture medium. Transcriptional analysis revealed that mtsA, encoding a metal-binding protein of an ABC transporter in S. pyogenes, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the perR mutant was more sensitive to the superoxide anion and had a reduced transcription of sodA, which encodes the sole superoxide dismutase of S. pyogenes. Complementation of the mutation with a functional perR gene restored 55Fe incorporation, response to peroxide stress, and transcription of both mtsA and sodA to levels comparable to those of wild-type bacteria. Finally, the perR mutant was attenuated in virulence in a murine air sac model of infection (P < 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of S. pyogenes.

scholarly journals Roles of Internal Cysteines in the Function, Localization, and Reactivity of the TraV Outer Membrane Lipoprotein Encoded by the F Plasmid

2002 ◽  
Vol 184 (11) ◽  
pp. 3126-3129 ◽  
Author(s):  
Robin L. Harris ◽  
Philip M. Silverman
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Osmotic Shock ◽  
Wild Type ◽  
F Plasmid ◽  
Outer Membrane Lipoprotein ◽  
Numerous Cell ◽  
Mixed Disulfides ◽  
Membrane Lipoprotein

ABSTRACT We have examined the functional role of two internal cysteine residues of the F-plasmid TraV outer membrane lipoprotein. Each was mutated to a serine separately and together to yield three mutant traV genes: traV C10S, traV C18S, and traV C10S/C18S. All three cysteine mutations complemented a traV mutant for DNA donor activity and for sensitivity to donor-specific bacteriophage; however, when measured by a transduction assay, the donor-specific DNA bacteriophage sensitivities of the traV C18S and, especially, traV C10S/C18S mutant strains were significantly less than those of the traV + and traV C10S strains. Thus, unlike the Agrobacterium tumefaciens T-plasmid-encoded VirB7 outer membrane lipoprotein, TraV does not require either internal cysteine to retain significant biological activity. By Western blot analysis, all three mutant TraV proteins were shown to accumulate in the outer membrane. However, by nonreducing gel electrophoresis, wild-type TraV and especially the TraVC18S mutant were shown to form mixed disulfides with numerous cell envelope proteins. This was not observed with the TraVC10S or TraVC10S/C18S proteins. Thus, it appears that TraV C10 is unusually reactive and that this reactivity is reduced by C18, perhaps by intramolecular oxidation. Finally, whereas the TraVC10S and TraVC18S proteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraVC10S/C18S protein was found in osmotic shock fluid and inner membrane fractions as well as outer membrane fractions. Hence, at least one cysteine is required for the efficient localization of TraV to the outer membrane.

scholarly journals Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites

2004 ◽  
Vol 380 (3) ◽  
pp. 805-813 ◽  
Author(s):  
Michael A. CATER ◽  
John FORBES ◽  
Sharon La FONTAINE ◽  
Diane COX ◽  
Julian F. B. MERCER
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Intracellular Trafficking ◽  
Copper Toxicity ◽  
Site Directed Mutagenesis ◽  
Metal Binding Sites ◽  
Wilson Protein ◽  
Necessary And Sufficient ◽  
Golgi Network

The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4–6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1–5 (amino acids 64–540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.

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