scholarly journals Regulation of Dual Activity of Ascorbate Peroxidase 1 From Arabidopsis thaliana by Conformational Changes and Posttranslational Modifications

2021 ◽  
Vol 12 ◽  
Author(s):  
Shubhpreet Kaur ◽  
Prapti Prakash ◽  
Dong-Ho Bak ◽  
Sung Hyun Hong ◽  
Chuloh Cho ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Ascorbate Peroxidase ◽  
Conformational Changes ◽  
Peroxidase Activity ◽  
Posttranslational Modifications ◽  
Negative Impact ◽  
Tyrosine Nitration ◽  
Ros Scavenging ◽  
Dimeric Form

Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS)-scavenging enzyme, which catalyzes the removal of hydrogen peroxide (H2O2) to prevent oxidative damage. The peroxidase activity of APX is regulated by posttranslational modifications (PTMs), such as S-nitrosylation, tyrosine nitration, and S-sulfhydration. In addition, it has been recently reported that APX functions as a molecular chaperone, protecting rice against heat stress. In this study, we attempted to identify the various functions of APX in Arabidopsis and the effects of PTMs on these functions. Cytosol type APX1 from Arabidopsis thaliana (AtAPX1) exists in multimeric forms ranging from dimeric to high-molecular-weight (HMW) complexes. Similar to the rice APX2, AtAPX1 plays a dual role behaving both as a regular peroxidase and a chaperone molecule. The dual activity of AtAPX1 was strongly related to its structural status. The main dimeric form of the AtAPX1 protein showed the highest peroxidase activity, whereas the HMW form exhibited the highest chaperone activity. Moreover, in vivo studies indicated that the structure of AtAPX1 was regulated by heat and salt stresses, with both involved in the association and dissociation of complexes, respectively. Additionally, we investigated the effects of S-nitrosylation, S-sulfhydration, and tyrosine nitration on the protein structure and functions using gel analysis and enzymatic activity assays. S-nitrosylation and S-sulfhydration positively regulated the peroxidase activity, whereas tyrosine nitration had a negative impact. However, no effects were observed on the chaperone function and the oligomeric status of AtAPX1. Our results will facilitate the understanding of the role and regulation of APX under abiotic stress and posttranslational modifications.

Melatonin synthesis enzymes interact with ascorbate peroxidase to protect against oxidative stress in cassava

2020 ◽  
Vol 71 (18) ◽  
pp. 5645-5655 ◽  
Author(s):  
Yujing Bai ◽  
Jingru Guo ◽  
Russel J Reiter ◽  
Yunxie Wei ◽  
Haitao Shi
Keyword(s):  
Oxidative Stress ◽  
Antioxidant Capacity ◽  
Stress Tolerance ◽  
Ascorbate Peroxidase ◽  
Stress Responses ◽  
Direct Interaction ◽  
Ros Scavenging ◽  
Melatonin Synthesis

Abstract Melatonin is an important indole amine hormone in animals and plants. The enzymes that catalyse melatonin synthesis positively regulate plant stress responses through modulation of the accumulation of reactive oxygen species (ROS). However, the relationship between melatonin biosynthetic enzymes and ROS-scavenging enzymes has not been characterized. In this study, we demonstrate that two enzymes of the melatonin synthesis pathway in Manihot esculenta (MeTDC2 and MeASMT2) directly interact with ascorbate peroxidase (MeAPX2) in both in vitro and in vivo experiments. Notably, in the presence of MeTDC2 and MeASMT2, MeAPX2 showed significantly higher activity and antioxidant capacity than the purified MeAPX2 protein alone. These findings indicate that MeTDC2–MeAPX2 and MeASMT2–MeAPX2 interactions both activate APX activity and increase antioxidant capacity. In addition, the combination of MeTDC2, MeASMT2, and MeAPX2 conferred improved resistance to hydrogen peroxide in Escherichia coli. Moreover, this combination also positively regulates oxidative stress tolerance in cassava. Taken together, these findings not only reveal a direct interaction between MeTDC2, MeASMT2, and MeAPX2, but also highlight the importance of this interaction in regulating redox homoeostasis and stress tolerance in cassava.

scholarly journals Cloning and Characterization of SCHIP-1, a Novel Protein Interacting Specifically with Spliced Isoforms and Naturally Occurring Mutant NF2 Proteins

2000 ◽  
Vol 20 (5) ◽  
pp. 1699-1712 ◽  
Author(s):  
Laurence Goutebroze ◽  
Estelle Brault ◽  
Christian Muchardt ◽  
Jacques Camonis ◽  
Gilles Thomas
Keyword(s):  
Tumor Suppressor ◽  
Conformational Changes ◽  
Posttranslational Modifications ◽  
Coiled Coil ◽  
Neurofibromatosis Type ◽  
Immunofluorescent Staining ◽  
Naturally Occurring ◽  
Cellular Context

ABSTRACT The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved in NF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members, implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as a tumor suppressor is unknown. In attempt to shed light on schwannomin function, we have identified a novel coiled-coil protein, SCHIP-1, that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein is homologous to human FEZ proteins and the relatedCaenorhabditis elegans gene product UNC-76. Immunofluorescent staining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmic membrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can be observed only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and 3, but not with the schwannomin isoform exhibiting growth-suppressive activity. Our observations suggest that SCHIP-1 interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslational modifications, alternative splicing, or mutations.

Conformation-dependent inactivation of tryptophan decarboxylase from Catharanthus roseus

1989 ◽  
Vol 67 (10) ◽  
pp. 730-734 ◽  
Author(s):  
J. A. Fernandez ◽  
W. G. W. Kurz ◽  
V. De Luca
Keyword(s):  
Conformational Changes ◽  
Catharanthus Roseus ◽  
Posttranslational Modifications ◽  
Half Life ◽  
Active Form ◽  
Tryptophan Decarboxylase ◽  
Monomeric Form ◽  
Conformational States ◽  
Dependent Inactivation

Catharanthus roseus tryptophan decarboxylase (TDC) was irreversibly inactivated in crude extracts and in purified form by incubation of the enzyme at 37 °C. The half-life of inactivation of TDC was approximately 0.75 h, whereas addition of Mn2+ or Mg2+ cations increased this half-life to 9.5 and to over 30 h, respectively. The evidence presented suggests that TDC could exist in vivo in equilibrium between a dimeric active form, which is stabilized by Mg2+ cations, and a monomeric form, which is susceptible to irreversible inactivation. When the equilibrium is shifted to monomer production and inactivation, TDC was quickly degraded by a proteolytic process, which could involve ATP. The results suggest that TDC activity may be regulated by posttranslational controls that shift the equilibrium between conformational states, some of which may be susceptible to irreversible inactivation followed by proteolysis.Key words: tryptophan decarboxylase, Mg2+-dependent conformational changes, posttranslational modifications.

scholarly journals In vivo protein tyrosine nitration in Arabidopsis thaliana

2011 ◽  
Vol 62 (10) ◽  
pp. 3501-3517 ◽  
Author(s):  
Jorge Lozano-Juste ◽  
Rosa Colom-Moreno ◽  
José León
Keyword(s):  
Arabidopsis Thaliana ◽  
Tyrosine Nitration ◽  
Protein Tyrosine ◽  
Protein Tyrosine Nitration

scholarly journals Methionine Sulfoxide Reductase B Regulates the Activity of Ascorbate Peroxidase of Banana Fruit

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 310
Author(s):  
Lu Xiao ◽  
Guoxiang Jiang ◽  
Huiling Yan ◽  
Hongmei Lai ◽  
Xinguo Su ◽  
...  
Keyword(s):  
Ascorbate Peroxidase ◽  
Posttranslational Modifications ◽  
Stress Responses ◽  
Redox State ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Banana Fruit ◽  
Compound I

Ascorbate peroxidase (APX) is a key antioxidant enzyme that is involved in diverse developmental and physiological process and stress responses by scavenging H2O2 in plants. APX itself is also subjected to multiple posttranslational modifications (PTMs). However, redox-mediated PTM of APX in plants remains poorly understood. Here, we identified and confirmed that MaAPX1 interacts with methionine sulfoxide reductase B2 (MsrB2) in bananas. Ectopic overexpression of MaAPX1 delays the detached leaf senescence induced by darkness in Arabidopsis. Sulfoxidation of MaAPX1, i.e., methionine oxidation, leads to loss of the activity, which is repaired partially by MaMsrB2. Moreover, mimicking sulfoxidation by mutating Met36 to Gln also decreases its activity in vitro and in vivo, whereas substitution of Met36 with Val36 to mimic the blocking of sulfoxidation has little effect on APX activity. Spectral analysis showed that mimicking sulfoxidation of Met36 hinders the formation of compound I, the first intermediate between APX and H2O2. Our findings demonstrate that the redox state of methionine in MaAPX1 is critical to its activity, and MaMsrB2 can regulate the redox state and activity of MaAPX1. Our results revealed a novel post-translational redox modification of APX.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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