Open research questions in Redox biology and oxidative stress

What the literature leaves open

• Moreover, the pharmacokinetic characteristics and long-term safety profiles of AZOX within the complex human tissue microenvironment remain to be systematically evaluated.

  Azoxystrobin induces mitochondrial dysfunction and mitochondrial pathway apoptosis by targeting the Prx1 Trp87 and Thr90 sites in oral leukoplakia · 2026 · DOI

• Tumor heterogeneity necessitates personalized therapeutic approaches. Lack of clinical validation for redox-targeted strategies. Context-aware therapeutic strategies should be developed.

  Redox-regulated cell death in gastric cancer: Molecular insights and therapeutic opportunities · 2026 · DOI

• Direct evidence for necroptosis as a major death pathway in gastric cancer in vivo remains limited. Most studies have relied on in vitro models or pharmacological inhibitors.

  Redox-regulated cell death in gastric cancer: Molecular insights and therapeutic opportunities · 2026 · DOI

• However, it remains unclear which proteins are carbonylated in human islets and how these modifications contribute to functional b-cell loss in T1D and T2D. Future studies are needed to determine whether FDA-approved drugs with carbonyl-scavenging properties, such as phenelzine, or pharmacological activators of NRF2, can effectively reduce protein carbonylation and preserve b-cell function in diabetes.

  Scars of oxidative stress: protein carbonylation and beta cell dysfunction in diabetes · 2025 · DOI

• Even though PKA is one of the most extensively studied eukaryotic protein kinases, critical knowledge gaps remain regarding the impact of oxPTMs on several aspects of its regulation, including holoenzyme conformation, kinase activation, substrate selection, and broader cellular functions. Likewise, the interplay between redox-dependent PKA regulation and classical cAMP-mediated activation is only beginning to be understood, yet this interplay is crucial for determining the overall effect of redox regulation on PKA function in several important cellular contexts. The susceptibility of both PKA regulatory and catalytic subunits to redox modification suggests a complex regulatory network that influences phosphorylation events underlying critical cellular activities, including cardiac contractility, lipogenesis, and inflammation. Therefore, while we begin to gain important insights into the mechanisms of crosstalk between redox- and PKA-dependent signaling, further research is needed to elucidate the physiological and pathological consequences of PKA oxidation. A deeper understanding of these mechanisms may offer new therapeutic targets for oxidative dysregulation of PKA in a variety of pervasive disorders, including cardiovascular disease, diabetes, and various neurological disorders. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life15040655/s1, Table S1: Select search terms used for literature review.; Table S2: Select physiological roles of PKA and associated disorders. Author Contributions: Conceptualization, E.S.E. and R.H.N.; investigation, E.S.E. and R.H.N.; writing—original draft preparation, E.S.E. and R.H.N.; writing—review and editing, M.F. and R.H.N.; supervision, M.F. and R.H.N.; project administration, R.H.N.; funding acquisition, M.F. and R.H.N. All authors have read and agreed to the published version of the manuscript. Life 2025, 15, 655 18 of 28 Funding: This research was funded by NIH/NIGMS, grant numbers 1R35GM153737 and 1SC1GM 130545, to R.H.N. and by NIH/NIHLBI, grant number 1R01HL162787, to M.F. The APC was funded by 1R35GM153737. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Acknowledgments: The authors would like to thank Bruce Freeman at the University of Pittsburgh School of Medicine and Leslie Poole at Wake Forest University School of Medicine for helpful discussions and support. Conflicts of Interest: The authors declare no conflict of interest.

  Redox Regulation of cAMP-Dependent Protein Kinase and Its Role in Health and Disease · 2025 · DOI

• 6.1. Redox-PTMs of Disease-Relevant Proteins to Diagnose NDDs There is a growing number of biofluid- and imaging-based biomarkers that have been proposed to help with the diagnosis of NDDs [228]. For instance, AD diagnosis cur- rently relies on a combination of clinical imaging and quantification of Aβ1-42, Tau protein and its phosphorylated form in the CSF of patients; however, these protein markers Antioxidants 2024, 13, 681 22 of 33 perform sub-optimally for early-stage sporadic AD. To tackle this, novel redox-modified biomarkers have been tested to help with the diagnosis of NDDs. For instance, Transthy- retin (TTR), which is an abundant protein in the CSF, is present under unmodified and oxidized forms, and the relative changes in unmodified vs oxidized forms have been shown by Nano-LC-ESI MS/MS to successfully discriminate people with AD and MCI as compared to controls when combined with levels of amyloid β1–42, tTau, pTau [207]. This study also highlighted the importance of optimising sample handling to avoid introduc- ing any artefactual redox-PTMs, in particular by controlling the duration of sample stor- age, experimental temperature, and the number of freeze–thaw cycles and developing a strict and reproducible sample preparation workflow [207]. After the initial phase of bi- omarker discovery in small- to medium-sized cohorts using untargeted proteomics, the biomarker validation phase is usually performed in larger independent cohorts. For in- stance, novel biomarkers to support the diagnosis of DLB (which can be clinically misdi- agnosed as AD) have been initially identified using label-free proteomics in CSF on a small cohort; this led to the discovery of a set of six potential biomarkers, including neurosecre- tory protein VGF [229]. This was later validated using targeted proteomics in an inde- pendent cohort of patients with DLB and in a larger cohort that included patients with AD, PD, or FTD [229]. In addition, multiple orthogonal proteomics methods have to be employed to validate new diagnosis biomarkers; these methods include targeted LC- MS/MS proteomics, ELISA, Olink proximity extension assay, and SomaScan aptamer pre- cipitation assay [229,230]. 6.2. Redox-PTMs of Disease-Relevant Proteins to Follow Disease Progression Given that ROS and RNS levels increase with NDDs progression, changes in redox- PTMs are expected over the course of the disease. For instance, an increase in the levels of S-glutathionylated GAPDH is observed in the blood of people living with AD and has been shown to be correlated with disease progression and severity [206]. Similarly, redox proteomics on postmortem brains from people on the AD continuum, from preclinical AD (PCAD), MCI, to AD, showed that nitration and carbonylation of proteins change with disease progression (reviewed in [231]): while no difference in global protein carbonyl or nitration

  Protein Oxidative Modifications in Neurodegenerative Diseases: From Advances in Detection and Modelling to Their Use as Disease Biomarkers · 2024 · DOI

• Vast and detailed knowledge of the Trx system in diverse organisms has accumulated during the past 50 years since the first description of thioredoxin in E. coli [74]. Currently, many aspects of their structure and reaction mechanisms involved in their catalytic and redox activities are well understood, including their compartment-specific functions and interacting partners. Notably, the advances in the mammalian Trx system have been paralleled by similar progress and detailed descriptions of the Trx system in model insect species, namely D. melanogaster and A. gambiae. Nevertheless, significant gaps still exist in our knowledge of the precise regulation of the Trx system within redox niches of intracellular compartments in connection with other components of the redox regulatory network. Furthermore, there is relatively limited knowledge of the interacting partners and target protein in insect cells compared to other organisms. Recent advances brought the implementation of new proteomic tools to charac- terise the Trx interactome in bacteria, plants and human cells [194–196]. It can be envisaged that applying these tools in insect studies would be feasible to provide more information to understand Trx function in insect development and stress responses. Genetically encoded redox biosensors have emerged as highly valued tools for the analysis of redox mechanisms at a subcellular resolution, as they can report the localisation and redox state of the target analyte based on changes in the fluorescence signal inten- sity [197]. The application of genetically encoded biosensors for analysing the glutathione redox potential in mitochondria revealed extensive crosstalk between the mitochondrial glutathione and thioredoxin systems and their decisive involvement in removing H2O2 produced in mitochondria [198]. The biosensor HyPer1, developed originally as an H2O2 probe, was utilised to obtain a deeper insight into the thioredoxin- and GSH-dependent reductive activity in cellular compartments [199]. Interestingly, the data suggest that Trx/TrxR predominantly reduces HyPer1 in the cytosol and nucleus, whereas GSH mainly reduces it in the mitochondria. As repeatedly described in this review, the efficient function of both Trx- and GSH-dependent redox control mechanisms are decisively dependent on the cellular sources of reduced cofactor NADPH as the source of reductive power. It is evident that new tools enabling the assessment of the NADP(H) redox status will be highly valuable. Recently, a novel genetically encoded ratiometric biosensor NERNST was reported to selectively monitor the NADP(H) redox potential in bacterial, plant and animal cells and organelles such as chloroplasts and mitochondria [200]. The adaptation and implementation of these molecular tools to study Trx and other redox systems in insect cell lines are yet to occur. Collectively, the accumulation of the recently achieved progress and newly available tools to study the Trx system within the context of the precise spatiotemporal redox regula- tions shows promise in advancing our understanding of the Trx system in insects. Future studies on its regulation and targets will uncover the emerging role of the Trx system beyond the antioxidant system in model insect species. Importantly, these advancements can be exploited to investigate the Trx system further in agronomically or ecologically relevant insect species, such as honey bees. Basic research on Trx and TrxR functions can be applied to novel concepts to increase bee health and their resistance to stress conditions induced by malnutrition, pathogens and environmental pollutants. Author Contributions: Conceptualization, A.G. and M.P.; writing—original draft preparation, A.G.; writing—review and editing, A.G. and M.P. All authors have read and agreed to the published version of the manuscript. Insects 2024, 15, 797 23 of 30 Funding: This research was funded by the Ministry of Education, Youth and Sport of the Czech Republic through the Inter-Action grant LUAUS24085. Data Availability Statement: No new data were created or analysed in this study. Data sharing is not applicable to this article. Conflicts of Interest: The authors declare no conflicts of interest.

  Thioredoxin System in Insects: Uncovering the Roles of Thioredoxins and Thioredoxin Reductase beyond the Antioxidant Defences · 2024 · DOI

• Macrophage polarization plays a critical role in parasite immune evasion, yet its regulatory mechanisms in cysticercosis have not been fully elucidated.

  TPx Protein of Cysticercus cellulosae Regulates Macrophage M2 Polarization via the cGMP-PKG Signaling Pathway · 2026 · DOI

• Whether AC itself ultimately advances to clinical application remains uncertain.

  Redox Remodeling as a Natural Product Strategy in Cancer Therapy: Lessons from Arnicolide C · 2026 · DOI

• Despite extensive research on the genome of chili peppers [73], the precise count of the GRX genes in this species remains unknown.

  Systematic Survey and Expression Analysis of the Glutaredoxin Gene Family in Capsicum annuum Under Hypoxia Stress · 2025 · DOI

• These findings support the conserved nature of OnTRX and provide a molecular basis for further investigation of its function in tilapia.

  The Role of Thioredoxin in Mitigating Ammonia-Induced Oxidative Stress in Nile Tilapia (Oreochromis niloticus) · 2026 · DOI

• The role of reactive oxygen species (ROS) in antibiotic-mediated bacterial killing and the emergence of resistance remains incompletely understood, particularly the identities and functional impacts of specific protein targets.

  Chemical proteomics reveals mechanisms of bacterial response to ROS mediated by antibiotics · 2026 · DOI

• Molecular mechanisms that control the growth of primary melanoma in the skin and promote progression are not fully understood.

  Thioredoxin interacting protein (TXNIP), a redox regulator, mediates the EPAC-RAP1 signaling dependency of primary melanoma · 2026 · DOI

• By reducing seizure recurrence, preserving neuronal integrity, and alleviating selected behavioral impairments, CB3 offers therapeutic benefits that extend beyond conventional ASMs and warrants further investigation for translation into clinical epilepsy treatment.

  Thioredoxin-mimetic peptide attenuates epilepsy progression and neurocognitive deficits · 2026 · DOI

• Existing redox proteomic methodologies are often limited by their efficiency, complexity, and cost.

  Cross-tissue deep profiling of redox proteome in obese mice using enhanced resin-assisted capture and data-independent acquisition · 2026 · DOI

• Hydrogen peroxide (H 2 O 2 ), a major mitochondrial ROS, significantly contributes to cardiotoxicity, yet its precise mechanistic role in DOX-induced cardiotoxicity remains incompletely understood.

  Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H2O2 detoxification · 2026 · DOI

• While environmental factors like light-dark transitions and high light modulate this pathway, the underlying molecular regulatory mechanisms remain unclear.

  Redox modulation of PGLP1 oligomerization mediated by thioredoxin f is crucial for plant responses to high light and fluctuating light · 2026 · DOI

• This phenotype indicates that these enzymes have a relevant function at early stages of plant development, although this function is poorly understood.

  2-Cys peroxiredoxins and the chaperone cpHSP70 act in concert in chloroplast biogenesis in Arabidopsis seedlings · 2026 · DOI

• Hypoxia promotes oral squamous cell carcinoma (OSCC) progression by disrupting redox equilibrium; however, how tumor cells precisely calibrate prosurvival reactive oxygen species levels remains unclear.

  Hypoxic Reprogramming of ACOX1-Driven HSP90AB1 Crotonylation Stabilizes Thioredoxin to Orchestrate Redox Homeostasis in Oral Squamous Cell Carcinoma · 2026 · DOI

• Despite its wide recognition across various biological systems, the full scope of oxytocin's activity remains incompletely understood.

  Metal- and Redox-Dependent Oxytocin Species Differentially Regulate Invasion and Migration in Triple-Negative Breast Cancer · 2026 · DOI

• Nevertheless, specific redox players involved in cell migration and invasion remain ill-defined.

  Redox regulation of cell migration via Nischarin S-glutathionylation · 2025 · DOI

• However, the precise regulatory networks governing ROS-mediated cancer cell death and their therapeutic applications remain inadequately characterized.

  Targeting oxidative stress-mediated regulated cell death as a vulnerability in cancer · 2025 · DOI

• The Golgi apparatus is an essential component of the secretory pathway, yet little is known about the concentration or redox state of GSH in this organelle.

  Characterization of the glutathione redox state in the Golgi apparatus · 2025 · DOI

• TRAP1 works as a homodimer, but recent evidence indicated that it forms tetramers whose effects remain elusive.

  Disulfide-mediated tetramerization of TRAP1 fosters its antioxidant and pro-neoplastic activities · 2025 · DOI

• While recent studies have highlighted the importance of redox balance in AML therapy, the specific contribution of protein redox signaling to resistance remains poorly understood.

  Redox-dependent protein S-glutathionylation governs azacitidine sensitivity and resistance in AML · 2025 · DOI

Most-cited papers in Redox biology and oxidative stress

• The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis · Nature · 1999 · 4,348 citations
• The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies · International Journal of Molecular Sciences · 2021 · 2,637 citations
• The Biology of Oxygen Radicals · Science · 1978 · 2,571 citations
• Oxidative Stress, Caloric Restriction, and Aging · Science · 1996 · 2,494 citations
• A Conserved Family of Prolyl-4-Hydroxylases That Modify HIF · Science · 2001 · 2,155 citations
• Protein Oxidation and Aging · Science · 1992 · 2,111 citations
• Prooxidant States and Tumor Promotion · Science · 1985 · 1,948 citations
• Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology · Nature Reviews Molecular Cell Biology · 2022 · 1,659 citations
• Redox metabolism: ROS as specific molecular regulators of cell signaling and function · Molecular Cell · 2021 · 1,245 citations
• The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas · Physiological Research · 2001 · 1,185 citations

Most recent work

• Oxidative Stress: Molecular Mechanisms, Diseases, and Therapeutic Targets · MedComm · 2026
• Thioredoxin-mimetic peptide attenuates epilepsy progression and neurocognitive deficits · Redox Biology · 2026
• The Role of Se-Containing Glutathione Peroxidases and Thioredoxin Reductases in Oncogenesis: Expression Paradoxes and Therapeutic Prospects · Antioxidants · 2026
• Chemical proteomics reveals mechanisms of bacterial response to ROS mediated by antibiotics · Free Radical Biology and Medicine · 2026
• Oxidized PDI promotes thrombus formation in oxidative stress · Blood · 2026
• A druggable redox switch on SHP1 controls macrophage inflammation · Nature Chemical Biology · 2026
• Beyond antioxidants: How ROS-RNS-RSS microdomains encode redox information in health and disease · Free Radical Biology and Medicine · 2026
• Making HEDS or tails of glutaredoxin catalysis: Direct reduction of bis(2-hydroxyethyl) disulfide as a non-glutathione disulfide substrate · Redox Biology · 2026
• What lies beyond thioredoxin reductase? Cyclometallated gold compounds reveal Sec selectivity in glutathione peroxidases · Free Radical Biology and Medicine · 2026
• Crosstalk of thioredoxin system and programmed cell death: from pathophysiology to novel therapy · Redox Biology · 2026

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