Nuclear-localized, iron-bound superoxide dismutase-2 antagonizes epithelial lineage programs to promote stemness of breast cancer cells via a histone demethylase activity.

The dichotomous behavior of superoxide dismutase-2 (SOD2) in cancer biology has long been acknowledged and more recently linked to different posttranslational forms of the enzyme. However, a distinctive activity underlying its tumor-promoting function is yet to be described. Here, we report that acetylation, one of such posttranslational modifications (PTMs), increases SOD2 affinity for iron, effectively changing the biochemical function of this enzyme from that of an antioxidant to a demethylase. Acetylated, iron-bound SOD2 localizes to the nucleus, promoting stem cell gene expression via removal of suppressive epigenetic marks such as H3K9me3 and H3K927me3. Particularly, H3K9me3 was specifically removed from regulatory regions upstream of Nanog and Oct-4, two pluripotency factors involved in cancer stem cell reprogramming. Phenotypically, cells expressing nucleus-targeted SOD2 (NLS-SOD2) have increased clonogenicity and metastatic potential. FeSOD2 operating as H3 demethylase requires H2O2 as substrate, which unlike cofactors of canonical demethylases (i.e., oxygen and 2-oxoglutarate), is more abundant in tumor cells than in normal tissue. Therefore, our results indicate that FeSOD2 is a demethylase with unique activities and functions in the promotion of cancer evolution toward metastatic phenotypes.

A mechanism by which gut microbiota elevates permeability and inflammation in obese/diabetic mice and human gut.

OBJECTIVE: Ample evidence exists for the role of abnormal gut microbiota composition and increased gut permeability ('leaky gut') in chronic inflammation that commonly co-occurs in the gut in both obesity and diabetes, yet the detailed mechanisms involved in this process have remained elusive. DESIGN: In this study, we substantiate the causal role of the gut microbiota by use of faecal conditioned media along with faecal microbiota transplantation. Using untargeted and comprehensive approaches, we discovered the mechanism by which the obese microbiota instigates gut permeability, inflammation and abnormalities in glucose metabolism. RESULTS: We demonstrated that the reduced capacity of the microbiota from both obese mice and humans to metabolise ethanolamine results in ethanolamine accumulation in the gut, accounting for induction of intestinal permeability. Elevated ethanolamine increased the expression of microRNA-miR-101a-3p by enhancing ARID3a binding on the miR promoter. Increased miR-101a-3p decreased the stability of zona occludens-1 (Zo1) mRNA, which in turn, weakened intestinal barriers and induced gut permeability, inflammation and abnormalities in glucose metabolism. Importantly, restoring ethanolamine-metabolising activity in gut microbiota using a novel probiotic therapy reduced elevated gut permeability, inflammation and abnormalities in glucose metabolism by correcting the ARID3a/miR-101a/Zo1 axis. CONCLUSION: Overall, we discovered that the reduced capacity of obese microbiota to metabolise ethanolamine instigates gut permeability, inflammation and glucose metabolic dysfunctions, and restoring ethanolamine-metabolising capacity by a novel probiotic therapy reverses these abnormalities. TRIAL REGISTRATION NUMBER: NCT02869659 and NCT03269032.

ABHD4 regulates adipocyte differentiation in vitro but does not affect adipose tissue lipid metabolism in mice.

Alpha/beta hydrolase domain-containing protein 4 (ABHD4) catalyzes the deacylation of N-acyl phosphatidyl-ethanolamine (NAPE) and lyso-NAPE to produce glycerophospho-N-acyl ethanolamine (GP-NAE). Through a variety of metabolic enzymes, NAPE, lyso-NAPE, and GP-NAE are ultimately converted into NAE, a group of bioactive lipids that control many physiological processes including inflammation, cognition, food intake, and lipolysis (i.e., oleoylethanolamide or OEA). In a diet-induced obese mouse model, adipose tissue Abhd4 gene expression positively correlated with adiposity. However, it is unknown whether Abhd4 is a causal or a reactive gene to obesity. To fill this knowledge gap, we generated an Abhd4 knockout (KO) 3T3-L1 pre-adipocyte. During adipogenic stimulation, Abhd4 KO pre-adipocytes had increased adipogenesis and lipid accumulation, suggesting Abhd4 is responding to (a reactive gene), not contributing to (not a causal gene), adiposity, and may serve as a mechanism for protecting against obesity. However, we did not observe any differences in adiposity and metabolic outcomes between whole-body Abhd4 KO or adipocyte-specific Abhd4 KO mice and their littermate control mice (both male and female) on chow or a high-fat diet. This might be because we found that deletion of Abhd4 did not affect NAE such as OEA production, even though Abhd4 was highly expressed in adipose tissue and correlated with fasting adipose OEA levels and lipolysis. These data suggest that ABHD4 regulates adipocyte differentiation in vitro but does not affect adipose tissue lipid metabolism in mice despite nutrient overload, possibly due to compensation from other NAPE and NAE metabolic enzymes.

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition.

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 µM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Quad-shot-immunotherapy: quad-shot radiotherapy with pembrolizumab for advanced/recurrent head and neck cancer.

Effective treatments for advanced/recurrent head and neck squamous-cell carcinoma are limited. For cases not curable by conventional local therapies, the immune checkpoint inhibitor pembrolizumab shows modest response rates. Quad-shot, a hypofractionated palliative radiotherapy regimen (14.8 Gy in four twice-daily fractions), can provide symptomatic relief, contributes to local control and may potentiate the effects of immune checkpoint inhibitors. In this study, 15 patients with advanced/recurrent head and neck squamous-cell carcinoma will be treated with pembrolizumab combined with up to three administrations of quad-shot before cycles four, eight and 13. Outcomes include disease response, survival and treatment toxicity. Correlative multiomics analysis of blood and saliva will identify molecular biomarkers of response to immune checkpoint inhibitor and the immune-related impact of quad-shot. Clinical trial registration: This study (WFBCCC 60320) is registered on NCT04454489 (ClinicalTrials.gov).

Advanced and recurrent head and neck cancers are difficult to treat. Most patients receive systemic therapies, such as chemotherapy or immunotherapy, with modest rates of cancer control. We aim to test the effectiveness of an immunotherapy drug called pembrolizumab in combination with a type of low-dose radiation therapy called quad-shot. Patients will receive pembrolizumab every 3 weeks and will be treated with one to three low-dose radiation therapy courses targeted at their cancer in the head and neck approximately every 12 weeks. We plan to measure how well the cancer responds to treatment, how long this response lasts, how long patients survive and treatment side effects.

Integrated Redox Proteomic Analysis Highlights New Mechanisms of Sensitivity to Silver Nanoparticles.

Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, but the molecular mechanisms underlying their activity remain elusive. In this study we profiled proteomics and redox proteomics changes induced by AgNPs in two lung cancer cell lines: AgNPs-sensitive Calu-1 and AgNPs-resistant NCI-H358. We show that AgNPs induce changes in protein abundance and reversible oxidation in a time and cell-line-dependent manner impacting critical cellular processes such as protein translation and modification, lipid metabolism, bioenergetics, and mitochondrial dynamics. Supporting confocal microscopy and transmission electron microscopy (TEM) data further emphasize mitochondria as a target of AgNPs toxicity differentially impacting mitochondrial networks and morphology in Calu-1 and NCI-H358 lung cells. Proteomics data are available via ProteomeXchange with identifier PXD021493.

Platelet-derived growth factor receptor beta activates Abl2 via direct binding and phosphorylation.

Abl family kinases are nonreceptor tyrosine kinases activated by diverse cellular stimuli that regulate cytoskeleton organization, morphogenesis, and adhesion. The catalytic activity of Abl family kinases is tightly regulated in cells by a complex set of intramolecular and intermolecular interactions and post-translational modifications. For example, the platelet-derived growth factor receptor beta (PDGFRß), important for cell proliferation and chemotaxis, is a potent activator of Abl family kinases. However, the molecular mechanism by which PDGFRß engages and activates Abl family kinases is not known. We show here that the Abl2 Src homology 2 domain directly binds to phosphotyrosine Y771 in the PDGFRß cytoplasmic domain. PDGFRß directly phosphorylates multiple novel sites on the N-terminal half of Abl2, including Y116, Y139, and Y161 within the Src homology 3 domain, and Y299, Y303, and Y310 on the kinase domain. Y116, Y161, Y272, and Y310 are all located at or near the Src homology 3/Src homology 2-kinase linker interface, which helps maintain Abl family kinases in an autoinhibited conformation. We also found that PDGFRß-mediated phosphorylation of Abl2 in vitro activates Abl2 kinase activity, but mutation of these four tyrosines (Y116, Y161, Y272, and Y310) to phenylalanine abrogated PDGFRß-mediated activation of Abl2. These findings reveal how PDGFRß engages and phosphorylates Abl2 leading to activation of the kinase, providing a framework to understand how growth factor receptors engage and activate Abl family kinases.

Antagonists targeting eEF2 kinase rescue multiple aspects of pathophysiology in Alzheimer's disease model mice.

It is imperative to develop novel therapeutic strategies for Alzheimer's disease (AD) and related dementia syndromes based on solid mechanistic studies. Maintenance of memory and synaptic plasticity relies on de novo protein synthesis, which is partially regulated by phosphorylation of eukaryotic elongation factor 2 (eEF2) via its kinase eEF2K. Abnormally increased eEF2 phosphorylation and impaired mRNA translation have been linked to AD. We recently reported that prenatal genetic suppression of eEF2K is able to prevent aging-related cognitive deficits in AD model mice, suggesting the therapeutic potential of targeting eEF2K/eEF2 signaling in AD. Here, we tested two structurally distinct small-molecule eEF2K inhibitors in two different lines of AD model mice after the onset of cognitive impairments. Our data revealed that treatment with eEF2K inhibitors improved AD-associated synaptic plasticity impairments and cognitive dysfunction, without altering brain amyloid ß (Aß) and tau pathology. Furthermore, eEF2K inhibition alleviated AD-associated defects in dendritic spine morphology, post-synaptic density formation, protein synthesis, and dendritic polyribosome assembly. Our results may offer critical therapeutic implications for AD, and the proof-of-principle study indicates translational implication of inhibiting eEF2K for AD and related dementia syndromes. Cover Image for this issue: https://doi.org/10.1111/jnc.15392.

Isoform-specific dysregulation of AMP-activated protein kinase signaling in a non-human primate model of Alzheimer's disease.

AMP-activated protein kinase (AMPK) is a molecular sensor that is critical for the maintenance of cellular energy homeostasis, disruption of which has been indicated in multiple neurodegenerative diseases including Alzheimer's disease (AD). Mammalian AMPK is a heterotrimeric complex and its enzymatic α subunit exists in two isoforms: AMPKα1 and AMPKα2. Here we took advantage of a recently characterized non-human primate (NHP) model with sporadic AD-like neuropathology to explore potential relationships between AMPK signaling and AD-like neuropathology. Subjects were nine female vervet monkeys aged 19.5 to 23.4 years old. Subjects were classified into three groups, control lacking AD pathology (n = 3), moderate AD pathology (n = 3), and more severe AD Pathology (n = 3). We found increased activity (assessed by phosphorylation) of AMPKα2 in hippocampi of NHP with AD-like neuropathology, compared to the subjects without AD pathology, with no alterations of AMPKα1 activity. Across all subjects, CSF Abeta42 was inversely associated with cerebral amyloid plaque density. Further, Aß plaque burden is correlated with levels of either soluble or insoluble brain Aß measurement. Unbiased mass spectrometry based proteomics studies combined with bioinformatics analysis revealed that many of the dysregulated proteins characteristic of AD neuropathology are associated with AMPK signaling. Our findings on the AMPK molecular signaling cascades provide further support for use of the NHP model to investigate new therapeutic strategies and development of novel biomarkers for Alzheimer's disease.

Protein expression alteration in hippocampus upon genetic repression of AMPKα isoforms.

The AMP-activated protein kinase (AMPK) is a molecular sensor to help maintain cellular energy homeostasis. AMPK is a heterotrimeric complex and its enzymatic catalytic subunit includes two isoforms: α1 and α2. Dysregulation of AMPK signaling is linked to neuronal diseases characterized with cognitive impairments. Emerging evidence also suggest isoform-specific roles of AMPK in the brain. AMPK regulates protein synthesis, which is critical for memory formation and neuronal plasticity. However, the consequence of altering AMPK activity on the translation of specific proteins in the brain is unknown. Here, we use unbiased mass spectrometry-based proteomics approach to analyze protein profile alterations in hippocampus and prefrontal cortex of transgenic mice in which the genes for the two AMPKα isoforms are conditionally deleted. The study revealed identities of proteins whose expression is sensitive to suppression of AMPKα1 and/or α2 isoform. These data may serve as a basis for future in-depth study. Elucidation of the functional relevance of the alteration of specific proteins could provide insights into identification of novel therapeutic targets for neuronal disorders characterized with AMPK signaling dysregulation and impaired cellular energy metabolism.

The mechanism of cell death induced by silver nanoparticles is distinct from silver cations.

BACKGROUND: Precisely how silver nanoparticles (AgNPs) kill mammalian cells still is not fully understood. It is not clear if AgNP-induced damage differs from silver cation (Ag+), nor is it known how AgNP damage is transmitted from cell membranes, including endosomes, to other organelles. Cells can differ in relative sensitivity to AgNPs or Ag+, which adds another layer of complexity to identifying specific mechanisms of action. Therefore, we determined if there were specific effects of AgNPs that differed from Ag+ in cells with high or low sensitivity to either toxicant. METHODS: Cells were exposed to intact AgNPs, Ag+, or defined mixtures of AgNPs with Ag+, and viability was assessed. The level of dissolved Ag+ in AgNP suspensions was determined using inductively coupled plasma mass spectrometry. Changes in reactive oxygen species following AgNP or Ag+ exposure were quantified, and treatment with catalase, an enzyme that catalyzes the decomposition of H2O2 to water and oxygen, was used to determine selectively the contribution of H2O2 to AgNP and Ag+ induced cell death. Lipid peroxides, formation of 4-hydroxynonenol protein adducts, protein thiol oxidation, protein aggregation, and activation of the integrated stress response after AgNP or Ag+ exposure were quantified. Lastly, cell membrane integrity and indications of apoptosis or necrosis in AgNP and Ag+ treated cells were examined by flow cytometry. RESULTS: We identified AgNPs with negligible Ag+ contamination. We found that SUM159 cells, which are a triple-negative breast cancer cell line, were more sensitive to AgNP exposure less sensitive to Ag+ compared to iMECs, an immortalized, breast epithelial cell line. This indicates that high sensitivity to AgNPs was not predictive of similar sensitivity to Ag+. Exposure to AgNPs increased protein thiol oxidation, misfolded proteins, and activation of the integrated stress response in AgNP sensitive SUM159 cells but not in iMEC cells. In contrast, Ag+ cause similar damage in Ag+ sensitive iMEC cells but not in SUM159 cells. Both Ag+ and AgNP exposure increased H2O2 levels; however, treatment with catalase rescued cells from Ag+ cytotoxicity but not from AgNPs. Instead, our data support a mechanism by which damage from AgNP exposure propagates through cells by generation of lipid peroxides, subsequent lipid peroxide mediated oxidation of proteins, and via generation of 4-hydroxynonenal (4-HNE) protein adducts. CONCLUSIONS: There are distinct differences in the responses of cells to AgNPs and Ag+. Specifically, AgNPs drive cell death through lipid peroxidation leading to proteotoxicity and necrotic cell death, whereas Ag+ increases H2O2, which drives oxidative stress and apoptotic cell death. This work identifies a previously unknown mechanism by which AgNPs kill mammalian cells that is not dependent upon the contribution of Ag+ released in extracellular media. Understanding precisely which factors drive the toxicity of AgNPs is essential for biomedical applications such as cancer therapy, and of importance to identifying consequences of unintended exposures.

Novel hyperoxidation resistance motifs in 2-Cys peroxiredoxins.

2-Cys peroxiredoxins (Prxs) modulate hydrogen peroxide (H2O2)-mediated cell signaling. At high H2O2 levels, eukaryotic Prxs can be inactivated by hyperoxidation and are classified as sensitive Prxs. In contrast, prokaryotic Prxs are categorized as being resistant to hyperoxidation and lack the GGLG and C-terminal YF motifs present in the sensitive Prxs. Additional molecular determinants that account for the subtle differences in the susceptibility to hyperoxidation remain to be identified. A comparison of a new, 2.15-Å-resolution crystal structure of Prx2 in the oxidized, disulfide-bonded state with the hyperoxidized structure of Prx2 and Prx1 in complex with sulfiredoxin revealed three structural regions that rearrange during catalysis. With these regions in hand, focused sequence analyses were performed comparing sensitive and resistant Prx groups. From this combinatorial approach, we discovered two novel hyperoxidation resistance motifs, motifs A and B, which were validated using mutagenesis of sensitive human Prxs and resistant Salmonella enterica serovar Typhimurium AhpC. Introduction and removal of these motifs, respectively, resulted in drastic changes in the sensitivity to hyperoxidation with Prx1 becoming 100-fold more resistant to hyperoxidation and AhpC becoming 800-fold more sensitive to hyperoxidation. The increased sensitivity of the latter AhpC variant was also confirmed in vivo These results support the function of motifs A and B as primary drivers for tuning the sensitivity of Prxs to different levels of H2O2, thus enabling the initiation of variable signaling or antioxidant responses in cells.

H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes.

Reactive oxygen species (ROS), in particular H2O2, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. H2O2 has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of H2O2 and inhibited by oxidation due to elevated levels of H2O2 The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 µm H2O2 Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSO2H) or sulfonic (RSO3H) acid. H2O2 treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to H2O2 inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on H2O2 levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular H2O2 rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell.

Endometrium and endometriosis tissue mitochondrial energy metabolism in a nonhuman primate model.

BACKGROUND: Endometriosis is the growth of uterine lining (endometrium) outside of the uterus. In other chronic inflammatory diseases, mitochondrial dysfunction is suspected of playing a role in disease pathogenesis. However, little is known about endometriosis mitochondrial function or its effects on tissue metabolism. The objectives of this study were to analyze mitochondrial function in nonhuman primate (NHP) endometrium and endometriosis tissue and to identify the metabolic features of these tissues that may contribute to disease. METHODS: Mitochondrial function in endometriosis tissue and endometrium was measured using mitochondrial respirometry analysis to determine if changes in oxidative phosphorylation exist in endometrium and endometriosis tissue compared to control endometrium from clinically healthy NHPs. Targeted metabolomics and multidimensional statistical analysis were applied to quantify key metabolites in energy and amino acid biosynthesis pathways. RESULTS: Mitochondrial respirometry assays showed endometrium from NHPs with endometriosis had reduced complex II-mediated oxygen consumption rates (OCR) across all energy states (basal, p = 0.01; state 3, p = 0.02; state 3u, p = 0.04; state 4o, p = 0.008) and endometriosis tissue had reduced state 3, complex I-mediated OCR (p = 0.02) and respiratory control rates (p = 0.01) compared to normal endometrium. Targeted metabolomics performed on tissue revealed carnitine (p = 0.001), creatine phosphate (p = 0.01), NADH (p = 0.0001), FAD (p = 0.001), tryptophan (p = 0.0009), and malic acid (p = 0.005) were decreased in endometriosis tissue compared to normal endometrium samples. FAD (p = 0.004), tryptophan (p = 0.0004) and malic acid (p = 0.03) were significantly decreased in endometrium from NHPs with endometriosis compared to normal endometrium. Significant metabolites identified in endometriosis and endometrium samples from animals with endometriosis were part of amino acid biosynthesis or energy metabolism pathways. CONCLUSIONS: Here, endometrial mitochondrial energy production and metabolism were decreased in endometrium and endometriosis tissue. Decreased mitochondrial energy production may be due to oxidative stress-induced damage to mitochondrial DNA or membranes, a shift in cell metabolism, or decreased energy substrate; however, the exact cause remains unknown. Additional research is needed to determine the implications of reduced mitochondrial energy production and metabolism on endometriosis and endometrium.

Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function.

Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M-1 s-1 and k = 5.99 M-1 s-1, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.

Mass Spectrometry in Advancement of Redox Precision Medicine.

Redox (portmanteau of reduction-oxidation) reactions involve the transfer of electrons between chemical species in biological processes fundamental to life. It is of outmost importance that cells maintain a healthy redox state by balancing the action of oxidants and antioxidants; failure to do so leads to a multitude of diseases including cancer, diabetes, fibrosis, autoimmune diseases, and cardiovascular and neurodegenerative diseases. From the perspective of precision medicine, it is therefore beneficial to interrogate the redox phenotype of the individual-similar to the use of genomic sequencing-in order to design tailored strategies for disease prevention and treatment. This chapter provides an overview of redox metabolism and focuses on how mass spectrometry (MS) can be applied to advance our knowledge in redox biology and precision medicine.

Decreased expression of pigment epithelium-derived factor within the penile tissues contributes to erectile dysfunction in diabetic rats.

Increased production of reactive oxygen species (ROS) and inflammation are major contributors to the development and progression of diabetes-associated erectile dysfunction (DMED). As an endogenous antioxidant and anti-inflammatory factor, the potential implication of pigment epithelium-derived factor (PEDF) in DMED has not been revealed. To assess the potential antioxidant and anti-inflammatory functions of PEDF in DMED, we first demonstrated that PEDF was significantly decreased at the levels of the mRNA and protein in the penis of diabetic rats compared with normal controls. To test the hypothesis that decreased the penile levels of PEDF are associated with oxidative stress and inflammation in DMED, an adenovirus expressing PEDF (Ad-PEDF) or the same titer of control virus (Ad-GFP) was intracavernously administered at 2 weeks after diabetic onset. After 6 weeks of treatment, we found that administration of Ad-PEDF could significantly increase erectile response to cavernosal nerve stimulation in the diabetic rats by restoring the endothelial NO synthase (eNOS), P-eNOS, and neuronal NO synthase (nNOS) protein levels to the standard levels represented in normal rats and by suppressing the levels of tumor necrosis factor-α (TNF-α) and oxidative stress. In conclusion, the present data indicated that the antioxidant and anti-inflammatory potential of PEDF plays important role in restoring erectile function by the inhibition of oxidative stress and TNF-α production.

Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes.

Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1-3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observedin situin human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism.

Crystal Structure and Substrate Specificity of Human Thioesterase 2: INSIGHTS INTO THE MOLECULAR BASIS FOR THE MODULATION OF FATTY ACID SYNTHASE.

The type I fatty acid synthase (FASN) is responsible for the de novo synthesis of palmitate. Chain length selection and release is performed by the C-terminal thioesterase domain (TE1). FASN expression is up-regulated in cancer, and its activity levels are controlled by gene dosage and transcriptional and post-translational mechanisms. In addition, the chain length of fatty acids produced by FASN is controlled by a type II thioesterase called TE2 (E.C. 3.1.2.14). TE2 has been implicated in breast cancer and generates a broad lipid distribution within milk. The molecular basis for the ability of the TE2 to compete with TE1 for the acyl chain attached to the acyl carrier protein (ACP) domain of FASN is unknown. Herein, we show that human TE1 efficiently hydrolyzes acyl-CoA substrate mimetics. In contrast, TE2 prefers an engineered human acyl-ACP substrate and readily releases short chain fatty acids from full-length FASN during turnover. The 2.8 Å crystal structure of TE2 reveals a novel capping domain insert within the α/ß hydrolase core. This domain is reminiscent of capping domains of type II thioesterases involved in polyketide synthesis. The structure also reveals that the capping domain had collapsed onto the active site containing the Ser-101-His-237-Asp-212 catalytic triad. This observation suggests that the capping domain opens to enable the ACP domain to dock and to place the acyl chain and 4'-phosphopantetheinyl-linker arm correctly for catalysis. Thus, the ability of TE2 to prematurely release fatty acids from FASN parallels the role of editing thioesterases involved in polyketide and non-ribosomal peptide synthase synthases.

Characterization of circulating APOL1 protein complexes in African Americans.

APOL1 gene renal-risk variants are associated with nephropathy and CVD in African Americans; however, little is known about the circulating APOL1 variant proteins which reportedly bind to HDL. We examined whether APOL1 G1 and G2 renal-risk variant serum concentrations or lipoprotein distributions differed from nonrisk G0 APOL1 in African Americans without nephropathy. Serum APOL1 protein concentrations were similar regardless of APOL1 genotype. In addition, serum APOL1 protein was bound to protein complexes in two nonoverlapping peaks, herein referred to as APOL1 complex A (12.2 nm diameter) and complex B (20.0 nm diameter). Neither of these protein complexes associated with HDL or LDL. Proteomic analysis revealed that complex A was composed of APOA1, haptoglobin-related protein (HPR), and complement C3, whereas complex B contained APOA1, HPR, IgM, and fibronectin. Serum HPR was less abundant on complex B in individuals with G1 and G2 renal-risk variant genotypes, relative to G0 (P = 0.0002-0.037). These circulating complexes may play roles in HDL metabolism and susceptibility to CVD.