Mitochondrial H2O2 release does not directly cause damage to chromosomal DNA.

Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of chromosomal DNA mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondria can directly damage nuclear DNA is largely lacking. In this study, we investigated the effects of H2O2 released by mitochondria or produced at the nucleosomes using a titratable chemogenetic approach. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2. Nuclear H2O2 gives rise to DNA damage and mutations and a subsequent p53 dependent cell cycle arrest. Mitochondrial H2O2 release shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. We conclude that H2O2 released from mitochondria is unlikely to directly damage nuclear genomic DNA, limiting its contribution to oncogenic transformation and aging.

FOXO transcription factors as mediators of stress adaptation.

The forkhead box protein O (FOXO, consisting of FOXO1, FOXO3, FOXO4 and FOXO6) transcription factors are the mammalian orthologues of Caenorhabditis elegans DAF-16, which gained notoriety for its capability to double lifespan in the absence of daf-2 (the gene encoding the worm insulin receptor homologue). Since then, research has provided many mechanistic details on FOXO regulation and FOXO activity. Furthermore, conditional knockout experiments have provided a wealth of data as to how FOXOs control development and homeostasis at the organ and organism levels. The lifespan-extending capabilities of DAF-16/FOXO are highly correlated with their ability to induce stress response pathways. Exogenous and endogenous stress, such as cellular redox stress, are considered the main drivers of the functional decline that characterizes ageing. Functional decline often manifests as disease, and decrease in FOXO activity indeed negatively impacts on major age-related diseases such as cancer and diabetes. In this context, the main function of FOXOs is considered to preserve cellular and organismal homeostasis, through regulation of stress response pathways. Paradoxically, the same FOXO-mediated responses can also aid the survival of dysfunctional cells once these eventually emerge. This general property to control stress responses may underlie the complex and less-evident roles of FOXOs in human lifespan as opposed to model organisms such as C. elegans.

Oxygen-consumption based quantification of chemogenetic H2O2 production in live human cells.

Reactive Oxygen Species (ROS) in the form of H2O2 can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H2O2 were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2 production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H2O2 formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H2O2. However, a method to directly quantify the amount of H2O2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2 production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, to estimate whether the ensuing level of H2O2 production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTERT cells, addition of 5 mM d-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H2O2. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H2O2 production to be able to discriminate the effects of H2O2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.

p53 Forms Redox-Dependent Protein-Protein Interactions through Cysteine 277.

Reversible cysteine oxidation plays an essential role in redox signaling by reversibly altering protein structure and function. Cysteine oxidation may lead to intra- and intermolecular disulfide formation, and the latter can drastically stabilize protein-protein interactions in a more oxidizing milieu. The activity of the tumor suppressor p53 is regulated at multiple levels, including various post-translational modification (PTM) and protein-protein interactions. In the past few decades, p53 has been shown to be a redox-sensitive protein, and undergoes reversible cysteine oxidation both in vitro and in vivo. It is not clear, however, whether p53 also forms intermolecular disulfides with interacting proteins and whether these redox-dependent interactions contribute to the regulation of p53. In the present study, by combining (co-)immunoprecipitation, quantitative mass spectrometry and Western blot we found that p53 forms disulfide-dependent interactions with several proteins under oxidizing conditions. Cysteine 277 is required for most of the disulfide-dependent interactions of p53, including those with 14-3-3θ and 53BP1. These interaction partners may play a role in fine-tuning p53 activity under oxidizing conditions.

DNA damage and oxidant stress activate p53 through differential upstream signaling pathways.

Stabilization and activation of the p53 tumor suppressor are triggered in response to various cellular stresses, including DNA damaging agents and elevated Reactive Oxygen Species (ROS) like H2O2. When cells are exposed to exogenously added H2O2, ATR/CHK1 and ATM/CHK2 dependent DNA damage signaling is switched on, suggesting that H2O2 induces both single and double strand breaks. These collective observations have resulted in the widely accepted model that oxidizing conditions lead to DNA damage that subsequently mediates a p53-dependent response like cell cycle arrest and apoptosis. However, H2O2 also induces signaling through stress-activated kinases (SAPK, e.g., JNK and p38 MAPK) that can activate p53. Here we dissect to what extent these pathways contribute to functional activation of p53 in response to oxidizing conditions. Collectively, our data suggest that p53 can be activated both by SAPK signaling and the DDR independently of each other, and which of these pathways is activated depends on the type of oxidant used. This implies that it could in principle be possible to modulate oxidative signaling to stimulate p53 without inducing collateral DNA damage, thereby limiting mutation accumulation in both healthy and tumor tissues.

The Human 2-Cys Peroxiredoxins form Widespread, Cysteine-Dependent- and Isoform-Specific Protein-Protein Interactions.

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.

Modulating organelle distribution using light-inducible heterodimerization in C. elegans.

The relative positioning of organelles underlies fundamental cellular processes, including signaling, polarization, and cellular growth. Here, we describe the usage of a light-dependent heterodimerization system, LOVpep-ePDZ, to alter organelle positioning locally and reversibly in order to study the functional consequences of organelle positioning. The protocol gives details on how to accomplish expression of fusion proteins encoding this system, describes the imaging parameters to achieve subcellular activation in C. elegans, and may be adapted for use in other model systems. For complete details on the use and execution of this protocol, please refer to De Henau et al. (2020).

A FOXO-dependent replication checkpoint restricts proliferation of damaged cells.

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Substantial accumulation of single-stranded DNA during DNA replication activates the DNA replication stress checkpoint response that slows progression from S/G2 to M phase to protect genomic integrity. Whether and how mild replication stress restricts proliferation remains controversial. Here, we identify a cell cycle exit mechanism that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the anaphase promoting complex/cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead box O (FOXO)-mediated inhibition of its transcription factor E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible and thereby play an important role in maintaining genomic stability.

Redox signaling modulates Rho activity and tissue contractility in the Caenorhabditis elegans spermatheca.

Actomyosin-based contractility in smooth muscle and nonmuscle cells is regulated by signaling through the small GTPase Rho and by calcium-activated pathways. We use the myoepithelial cells of the Caenorhabditis elegans spermatheca to study the mechanisms of coordinated myosin activation in vivo. Here, we show that redox signaling modulates RHO-1/Rho activity in this contractile tissue. Exogenously added as well as endogenously generated hydrogen peroxide decreases spermathecal contractility by inhibition of RHO-1, which depends on a conserved cysteine in its nucleotide binding site (C20). Further, we identify an endogenous gradient of H2O2 across the spermathecal tissue, which depends on the activity of cytosolic superoxide dismutase, SOD-1. Collectively, we show that SOD-1-mediated H2O2 production regulates the redox environment and fine tunes Rho activity across the spermatheca through oxidation of RHO-1 C20.

Cross-talk between redox signalling and protein aggregation.

It is well established that both an increase in reactive oxygen species (ROS: i.e. O2•-, H2O2 and OH•), as well as protein aggregation, accompany ageing and proteinopathies such as Parkinson's and Alzheimer's disease. However, it is far from clear whether there is a causal relation between the two. This review describes how protein aggregation can be affected both by redox signalling (downstream of H2O2), as well as by ROS-induced damage, and aims to give an overview of the current knowledge of how redox signalling affects protein aggregation and vice versa. Redox signalling has been shown to play roles in almost every step of protein aggregation and amyloid formation, from aggregation initiation to the rapid oligomerization of large amyloids, which tend to be less toxic than oligomeric prefibrillar aggregates. We explore the hypothesis that age-associated elevated ROS production could be part of a redox signalling-dependent-stress response in an attempt to curb protein aggregation and minimize toxicity.

Mitochondria-Derived H2O2 Promotes Symmetry Breaking of the C. elegans Zygote.

Symmetry breaking is an essential step in cell differentiation and early embryonic development. However, the molecular cues that trigger symmetry breaking remain largely unknown. Here, we show that mitochondrial H2O2 acts as a symmetry-breaking cue in the C. elegans zygote. We find that symmetry breaking is marked by a local H2O2 increase and coincides with a relocation of mitochondria to the cell cortex. Lowering endogenous H2O2 levels delays the onset of symmetry breaking, while artificially targeting mitochondria to the cellular cortex using a light-induced heterodimerization technique is sufficient to initiate symmetry breaking in a H2O2-dependent manner. In wild-type development, both sperm and maternal mitochondria contribute to symmetry breaking. Our findings reveal that mitochondrial H2O2-signaling promotes the onset of polarization, a fundamental process in development and cell differentiation, and this is achieved by both mitochondrial redistribution and differential H2O2-production.

Reactive Oxygen Species Induced p53 Activation: DNA Damage, Redox Signaling, or Both?

Significance: The p53 tumor suppressor has been dubbed the "guardian of genome" because of its various roles in the response to DNA damage such as DNA damage repair, cell cycle arrest, senescence, and apoptosis, all of which are in place to prevent mutations from being passed on down the lineage. Recent Advances: Reactive oxygen species (ROS), for instance hydrogen peroxide derived from mitochondrial respiration, have long been regarded mainly as a major source of cellular damage to DNA and other macromolecules. Critical Issues: More recently, ROS have been shown to also play important physiological roles as second messengers in so-called redox signaling. It is, therefore, not clear whether the observed activation of p53 by ROS is mediated through the DNA damage response, redox signaling, or both. In this review, we will discuss the similarities and differences between p53 activation in response to DNA damage and redox signaling in terms of upstream signaling and downstream transcriptional program activation. Future Directions: Understanding whether and how DNA damage and redox signaling-dependent p53 activation can be dissected could be useful to develop anti-cancer therapeutic p53-reactivation strategies that do not depend on the induction of DNA damage and the resulting additional mutational load.

Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16INK4A.

The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-ß sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression.

FOXO transcription factors are regulators of cellular homeostasis and putative tumor suppressors, yet the role of FOXO in cancer progression remains to be determined. The data on FOXO function, particularly for epithelial cancers, are fragmentary and come from studies that focused on isolated aspects of cancer. To clarify the role of FOXO in epithelial cancer progression, we characterized the effects of inducible FOXO activation and loss in a mouse model of metastatic invasive lobular carcinoma. Strikingly, either activation or loss of FOXO function suppressed tumor growth and metastasis. We show that the multitude of cellular processes critically affected by FOXO function include proliferation, survival, redox homeostasis, and PI3K signaling, all of which must be carefully balanced for tumor cells to thrive.Significance: FOXO proteins are not solely tumor suppressors, but also support tumor growth and metastasis by regulating a multitude of cellular processes essential for tumorigenesis. Cancer Res; 78(9); 2356-69. ©2018 AACR.

Proteome-wide Changes in Protein Turnover Rates in C. elegans Models of Longevity and Age-Related Disease.

The balance between protein synthesis and protein breakdown is a major determinant of protein homeostasis, and loss of protein homeostasis is one of the hallmarks of aging. Here we describe pulsed SILAC-based experiments to estimate proteome-wide turnover rates of individual proteins. We applied this method to determine protein turnover rates in Caenorhabditis elegans models of longevity and Parkinson's disease, using both developing and adult animals. Whereas protein turnover in developing, long-lived daf-2(e1370) worms is about 30% slower than in controls, the opposite was observed in day 5 adult worms, in which protein turnover in the daf-2(e1370) mutant is twice as fast as in controls. In the Parkinson's model, protein turnover is reduced proportionally over the entire proteome, suggesting that the protein homeostasis network has a strong ability to adapt. The findings shed light on the relationship between protein turnover and healthy aging.

The Hallmarks of Cancer from a Redox Perspective.

SIGNIFICANCE: For a healthy cell to turn into a cancer cell and grow out to become a tumor, it needs to undergo a series of complex changes and acquire certain traits, summarized as "The Hallmarks of Cancer." These hallmarks can all be regarded as the result of altered signal transduction cascades and an understanding of these cascades is essential for cancer treatment. RECENT ADVANCES: Redox signaling is a long overlooked form of signal transduction that proceeds through the reversible oxidation of cysteines in proteins and that uses hydrogen peroxide as a second messenger. CRITICAL ISSUES: In this article, we provide examples that show that redox signaling is involved in the regulation of proteins and signaling cascades that play roles in every hallmark of cancer. FUTURE DIRECTIONS: An understanding of how redox signaling and "classical" signal transduction are intertwined could hold promising strategies for cancer therapy in the future. Antioxid. Redox Signal. 25, 300-325.

Covalent targeting of acquired cysteines in cancer.

The thiolate side chain of cysteine has a unique functionality that drug hunters and chemical biologists have begun to exploit. For example, targeting cysteine residues in the ATP-binding pockets of kinases with thiol-reactive molecules has afforded increased selectivity and potency to drugs like imbrutinib, which inhibits the oncogene BTK, and CO-1686 and AZD9291 that target oncogenic mutant EGFR. Recently, disulfide libraries and targeted GDP-mimetics have been used to selectively label the G12C oncogenic mutation in KRAS. We reasoned that other oncogenes contain mutations to cysteine, and thus screened the Catalog of Somatic Mutations in Cancer for frequently acquired cysteines. Here, we describe the most common mutations and discuss how these mutations could be potential targets for cysteine-directed personalized therapeutics.