scholarly journals Stepwise Oxidations Play Key Roles in the Structural and Functional Regulations of DJ-1

2021 ◽  
Author(s):  
In-Kang Song ◽  
Mi-Sun Kim ◽  
James E Ferrell Jr ◽  
Dong-Hae Shin ◽  
Kong-Joo Lee
Keyword(s):  
Mass Spectrometry ◽  
Structural Integrity ◽  
Antioxidant Activities ◽  
Neuronal Cell ◽  
Cysteine Residue ◽  
Disulfide Crosslinking ◽  
Functional Changes ◽  
Exchange Mass Spectrometry ◽  
Antioxidant Protein ◽  
Dopaminergic Neuronal Cell

DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. Human DJ-1 has three cysteine residues (Cys46, Cys53 and Cys106). We found that, in addition to Cys106, Cys46 is the most reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe that selectively reacts with redox sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with adjacent resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. Mutants (C46A and C53A), not forming Cys46-Cys53 disulfide crosslinking, increased oxidation of Cys106 to sulfinic and sulfonic acids. Furthermore, we found that DJ-1 C46A mutant has distorted unstable structure identified by biochemical assay and employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike wild type DJ-1. These findings suggest that all three Cys residues including Cys46-Cys53 disulfide crosslinking are required for maintaining the structural integrity, the regulation process and cellular function as an antioxidant protein. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

scholarly journals Stepwise Oxidations Play Key Roles in the Structural and Functional Regulations of DJ-1

2021 ◽  
Author(s):  
In-Kang Song ◽  
Mi-Sun Kim ◽  
James E. Ferrell ◽  
Dong-Hae Shin ◽  
Kong-Joo Lee
Keyword(s):  
Mass Spectrometry ◽  
Disulfide Bond ◽  
Antioxidant Activities ◽  
Neuronal Cell ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Disulfide Crosslinking ◽  
Functional Changes ◽  
Exchange Mass Spectrometry ◽  
Dopaminergic Neuronal Cell

AbstractDJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. We found that Cys46 is also reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe that selectively reacts with redox sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. We also found that Cys46-Cys53 disulfide crosslinking affects the oxidative state of the third Cys106, which shows the crosstalk among three cysteine residues of DJ-1. Furthermore, we demonstrated that DJ-1 C46A mutant, not forming Cys46-Cys53 intra-disulfide bond, lost structural stability of DJ-1 employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike wild type DJ-1. These findings suggest that DJ-1 regulates its structure and activities by concerted oxidative modifications of three cysteine residues. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

scholarly journals Labile disulfide bonds are common at the leucocyte cell surface

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110010 ◽  
Author(s):  
Clive Metcalfe ◽  
Peter Cresswell ◽  
Laura Ciaccia ◽  
Benjamin Thomas ◽  
A. Neil Barclay
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Disulfide Bonds ◽  
Reduction Process ◽  
Cysteine Residue ◽  
Surface Proteins ◽  
Protein Activity ◽  
Functional Changes ◽  
Reducing Conditions

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.

scholarly journals Unraveling cardiolipin-induced conformational change of cytochrome c through H/D exchange mass spectrometry and quartz crystal microbalance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sin-Cih Sun ◽  
Hung-Wei Huang ◽  
Yi-Ting Lo ◽  
Min-Chieh Chuang ◽  
Yuan-Hao Howard Hsu
Keyword(s):  
Mass Spectrometry ◽  
Quartz Crystal Microbalance ◽  
Quartz Crystal ◽  
Interaction Model ◽  
Moderate Increase ◽  
Exchange Mass Spectrometry ◽  
Mitochondrial Regulation ◽  
Crucial Component ◽  
Drastic Increase ◽  
Hydrogen Deuterium

AbstractCardiolipin (CL), a crucial component in inner mitochondrial membranes, interacts with cytochrome c (cyt c) to form a peroxidase complex for the catalysis of CL oxidation. Such interaction is pivotal to the mitochondrial regulation of apoptosis and is affected by the redox state of cyt c. In the present study, the redox-dependent interaction of cyt c with CL was investigated through amide hydrogen/deuterium exchange coupled with mass spectrometry (HDXMS) and quartz crystal microbalance with dissipation monitoring (QCM-D). Ferrous cyt c exhibited a more compact conformation compared with its ferric form, which was supported by the lower number of deuterons accumulated and the greater amplitude reduction on dissipation. Upon association with CL, ferrous cyt c resulted in a moderate increase in deuteration, whereas the ferric form caused a drastic increase of deuteration, which indicated that CL-bound ferric cyt c formed an extended conformation. These results were consistent with those of the frequency (f) − dissipation (D) experiments, which revealed that ferric cyt c yielded greater values of |ΔD/Δf| within the first minute. Further fragmentation analysis based on HDXMS indicated that the effect of CL binding was considerably different on ferric and ferrous cyt c in the C-helix and the Loop 9–24. In ferric cyt c, CL binding affected Met80 and destabilized His18 interaction with heme, which was not observed with ferrous cyt c. An interaction model was proposed to explain the aforementioned results.

Hydrogen/Deuterium and 16O/18O-Exchange Mass Spectrometry Boosting the Reliability of Compound Identification

2020 ◽  
Vol 92 (10) ◽  
pp. 6877-6885 ◽  
Author(s):  
Yury Kostyukevich ◽  
Alexander Zherebker ◽  
Alexey Orlov ◽  
Oxana Kovaleva ◽  
Tatyana Burykina ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Compound Identification ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium
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