Enzymology
Oxidative stress–induced autonomous activation of the calcium/calmodulin-dependent kinase II involves disulfide formation in the regulatory domain
Calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ) has a pivotal role in cardiac signaling. Constitutive and deleterious CaMKII “autonomous” activation is induced by oxidative stress, and the previously reported mechanism involves oxidation of methionine residues in the regulatory domain. Here, we demonstrate that covalent oxidation leads to a disulfide bond with Cys273 in the regulatory domain causing autonomous activity. Autonomous activation was induced by treating CaMKII with diamide or histamine chloramine, two thiol-oxidizing agents.The ChaC family of γ-glutamyl cyclotransferases is required for Leishmania to switch to a slow growth state and for long-term survival of the parasite
The ChaC family of γ-glutamyl cyclotransferases is conserved throughout all Kingdoms and catalyzes the degradation of GSH. So far, the ChaC family proteins in trypanosomal parasites are missing in the literature. Here, we report two members of the ChaC family of γ-glutamyl cyclotransferases (LmChaC2a and LmChaC2b) in the unicellular pathogen Leishmania. Activity measurements suggest that these proteins catalyze degradation of GSH but no other γ-glutamyl peptides. Recombinant LmChaC2a protein shows ∼17-fold lower catalytic efficiency (kcat ∼ 0.9 s−1) than LmChaC2b (kcat ∼ 15 s−1), although they showed comparable Km values (∼1.75 mM for LmChaC2a and ∼2.0 mM for LmChaC2b) toward GSH.The role of cysteines in the structure and function of OGG1
8-Oxoguanine glycosylase (OGG1) is a base excision repair enzyme responsible for the recognition and removal of 8-oxoguanine, a commonly occurring oxidized DNA modification. OGG1 prevents the accumulation of mutations and regulates the transcription of various oxidative stress–response genes. In addition to targeting DNA, oxidative stress can affect proteins like OGG1 itself, specifically at cysteine residues. Previous work has shown that the function of OGG1 is sensitive to oxidants, with the cysteine residues of OGG1 being the most likely site of oxidation.Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase
Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated.Structure-function analyses of alkylhydroperoxidase D from Streptococcus pneumoniae reveal an unusual three-cysteine active site architecture
During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo. We demonstrate here that SpAhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, SpAhpD does not mediate electron transfer to SpAhpC.Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens
Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized.Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA
The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA.Angiogenin activates the astrocytic Nrf2/antioxidant-response element pathway and thereby protects murine neurons from oxidative stress
The angiogenin (ANG) gene is mutated frequently in individuals with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Delivering human ANG to mice that display ALS-like symptoms extends their lifespan and improves motor function. ANG is a secretory vertebrate RNase that enters neuronal cells and cleaves a subset of tRNAs, leading to the inhibition of translation initiation and the assembly of stress granules. Here, using murine neuronal and astrocytic cell lines, we find that ANG triggers the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway, which provides a critical cellular defense against oxidative stress.Isotopic tagging of oxidized and reduced cysteines (iTORC) for detecting and quantifying sulfenic acids, disulfides, and free thiols in cells
Oxidative modifications of cysteine residues are an important component in signaling pathways, enzymatic regulation, and redox homeostasis. Current direct and indirect methods detect specific modifications and a general binary population of “free” or “oxidized” cysteines, respectively. In an effort to combine both direct and indirect detection strategies, here we developed a method that we designate isotopic tagging of oxidized and reduced cysteines (iTORC). This method uses synthetic molecules for rapid isotopic coding of sulfenic acids, reduced cysteines, and disulfides in cells.Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I
Site-specific suppressors of superoxide production (named S1QELs) in the quinone-reaction site in mitochondrial respiratory complex I during reverse electron transfer have been previously reported; however, their mechanism of action remains elusive. Using bovine heart submitochondrial particles, we herein investigated the effects of S1QELs on complex I functions. We found that the inhibitory effects of S1QELs on complex I are distinctly different from those of other known quinone-site inhibitors.Reversible S-glutathionylation of human 6-pyruvoyl tetrahydropterin synthase protects its enzymatic activity
6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions.Myristoylated methionine sulfoxide reductase A is a late endosomal protein
Methionine residues in proteins provide antioxidant defense by reacting with oxidizing species, which oxidize methionine to methionine sulfoxide. Reduction of the sulfoxide back to methionine is catalyzed by methionine sulfoxide reductases, essential for protection against oxidative stress. The nonmyristoylated form of methionine sulfoxide reductase A (MSRA) is present in mitochondria, whereas the myristoylated form has been previously reported to be cytosolic. Despite the importance of MSRA in antioxidant defense, its in vivo binding partners and substrates have not been identified.The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis
The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress.Heme–thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition
Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear.Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction
Human RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts.Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine
Numerous ribonucleotides are incorporated into the genome during DNA replication. Oxidized ribonucleotides can also be erroneously incorporated into DNA. Embedded ribonucleotides destabilize the structure of DNA and retard DNA synthesis by DNA polymerases (pols), leading to genomic instability. Mammalian cells possess translesion DNA synthesis (TLS) pols that bypass DNA damage. The mechanism of TLS and repair of oxidized ribonucleotides remains to be elucidated. To address this, we analyzed the miscoding properties of the ribonucleotides riboguanosine (rG) and 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG) during TLS catalyzed by the human TLS pols κ and η in vitro.Chondroitin 4-O-Sulfotransferase Is Indispensable for Sulfation of Chondroitin and Plays an Important Role in Maintaining Normal Life Span and Oxidative Stress Responses in Nematodes
Chondroitin sulfate (CS)/chondroitin (Chn) chains are indispensable for embryonic cell division and cytokinesis in the early developmental stages in Caenorhabditis elegans and mice, whereas heparan sulfate (HS) is essential for axon guidance during nervous system development. These data indicate that the fundamental functions of CS and HS are conserved from worms to mammals and that the function of CS/Chn differs from that of HS. Although previous studies have shown that C. elegans produces HS and non-sulfated Chn, whether the organism produces CS remains unclear.Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases
The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBankTM and PDB databases, but the enzyme was not kinetically characterized. In this study, PA1024 was purified to high levels, and the enzymatic activity was investigated by spectroscopic and polarographic techniques. Purified PA1024 did not exhibit nitronate monooxygenase activity; however, it displayed NADH:quinone reductase and a small NADH:oxidase activity.Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis
Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys57 and Cys146, which have been linked to protein stability and folding via forming a disulfide bond, and Cys6 and Cys111 as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins.Targeting Thioredoxin Reductase by Parthenolide Contributes to Inducing Apoptosis of HeLa Cells
Parthenolide (PTL), a major active sesquiterpene lactone from the herbal plant Tanacetum parthenium, has been applied in traditional Chinese medicine for centuries. Although PTL demonstrates potent anticancer efficacy in numerous types of malignant cells, the cellular targets of PTL have not been well defined. We reported here that PTL interacts with both cytosolic thioredoxin reductase (TrxR1) and mitochondrial thioredoxin reductase (TrxR2), two ubiquitous selenocysteine-containing antioxidant enzymes, to elicit reactive oxygen species-mediated apoptosis in HeLa cells.Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems
Protein modification by small ubiquitin-related modifiers (SUMOs) is essential and conserved in the malaria parasite, Plasmodium falciparum. We have previously shown that interactions between the SUMO E1-activating and E2-conjugating enzyme in P. falciparum are distinct compared with human, suggesting a potential target for development of parasite-specific inhibitors of SUMOylation. The parasite asexual trophozoite stage is susceptible to iron-induced oxidative stress and is subsequently a target for many of the current anti-malarial drugs.A Novel Role of Proline Oxidase in HIV-1 Envelope Glycoprotein-induced Neuronal Autophagy
Background: Stress response autophagy is induced during HIV-1 glycoprotein “gp120”-mediated neurotoxicity. However, the underlying mechanisms are poorly understood.Results: HIV-1 gp120 induces proline oxidase that elicits ROS-mediated neuronal autophagy.Conclusion: Protective autophagy during HIV-1 gp120 neurotoxicity is partly dependent on proline oxidase-induced ROS.Significance: This is the first report that demonstrates the functional role of proline oxidase in HIV-1 gp120-mediated neuronal autophagy.CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage
Background: CUX2 contains three Cut repeat domains and is expressed in postmitotic neurons.Results: Cut repeats stimulate the OGG1 DNA glycosylase, and lower or higher CUX2 expression, respectively, delays or accelerates repair of oxidative DNA damage.Conclusion: CUX2 functions as an accessory factor in base excision repair.Significance: CUX2 contributes to the maintenance of genome integrity in long lived neurons.Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity
Background: Oxidative stress leads to increased PRMT1 expression and ADMA accumulation.Results: PRMT1 activity is increased by reductants and decreased by oxidants.Conclusion: PRMT1 activity is regulated in a redox-sensitive manner.Significance: The role of PRMT1 activity in the oxidative stress response may be more complex than previously thought.
