Case 7 - BBS2042

Case 7 - Oxidative Stress

1. Learning Goals

  • Understand the following:

    • Redox reactions

    • Physiological processes where redox is important

    • Redox production in normal physiological conditions

    • Redox signalling in normal physiological conditions:

    • Changes in gene transcription (KEAP1 and NRF2)

    • Redox balance by antioxidants (enzymatic and non-enzymatic)

    • Reactive Oxygen Species (ROS) as a messenger in cell signaling (reversible and non-reversible effects):

    • Cysteine oxidation

    • Homeostasis disruption by redox imbalance:

    • Alzheimer's

    • Cancer

    • Cardiovascular disease

    • Treatment of diseases caused by redox imbalance by antioxidants:

    • Is it possible and effective? Arguments for and against

2. What is a Redox Reaction?

  • Definition: A redox reaction is a chemical reaction involving the transfer of electrons between molecules.

    • Oxidation: Loss of electrons.

    • Reduction: Gain of electrons.

3. Production of Reactive Oxygen Species (ROS)

3.1 Mitochondrial Electron Transport Chain (Main Source)

  • Overview: The mitochondria are the primary source of ROS during cellular respiration.

    • In the Electron Transport Chain (ETC), electrons are transferred through complexes I–IV, reducing oxygen to water.

    • A small percentage of electrons leak prematurely, mainly from Complex I and Complex III.

    • Reaction:
      O2 + e^- \rightarrow O2^{•-} (this forms superoxide, $O_2^{•-}$).

  • Further Conversions:

    • Superoxide can be converted into other ROS:

    • Superoxide dismutase (SOD) converts it into hydrogen peroxide ($H2O2$).

    • Hydrogen peroxide ($H2O2$) can further form hydroxyl radicals ($•OH$) through reactions such as the Fenton reaction with iron.

3.2 NADPH Oxidase (ROS for Signalling and Immunity)

  • Certain cells intentionally produce ROS using the enzyme NADPH oxidase (NOX).

    • Found especially in immune cells (neutrophils, macrophages).

  • Mechanism: Transfers electrons from NADPH to oxygen, generating superoxide.

    • Importance:

    • Pathogen killing during the respiratory burst.

    • Redox signalling in various cell types.

3.3 Other Enzymatic Sources

  • Several other enzymes also generate ROS as part of their catalytic activity:

    • Xanthine oxidase: Produces superoxide during purine metabolism.

    • Cytochrome P450 enzymes: Can leak electrons during detoxification reactions.

    • Monoamine oxidase (MAO): Produces $H2O2$ during neurotransmitter breakdown.

    • Peroxisomal oxidases: Generate $H2O2$ during fatty acid oxidation.

3.4 External or Cellular Stress Sources

  • ROS production can increase due to external factors:

    • UV radiation

    • Ionizing radiation

    • Inflammation

    • Environmental toxins

    • Ischemia–reperfusion injury

  • These conditions often amplify mitochondrial or enzymatic ROS production.

4. Physiological Processes

4.1 Cell Signalling and Signal Transduction

  • ROS act as second messengers in many signalling pathways.

    • Hydrogen peroxide ($H2O2$) can reversibly oxidize cysteine residues in proteins, modifying the activity of signalling proteins such as kinases and phosphatases.

  • ROS often inhibit protein tyrosine phosphatases, prolonging phosphorylation signals.

  • Processes influenced include:

    • Growth factor signalling

    • Hormone signalling

    • Cytokine signalling

  • Important pathways regulated by ROS include:

    • MAPK signalling

    • PI3K/Akt signalling

    • NF-κB signalling

4.2 Immune Defense (Respiratory Burst)

  • ROS are essential for killing pathogens during immune responses:

    • Neutrophils and macrophages produce ROS via NADPH oxidase.

    • This process is known as the respiratory burst.

    • Superoxide and other ROS damage bacterial membranes, DNA, and proteins, which is critical for innate immunity.

4.3 Regulation of Gene Expression

  • ROS regulate transcription factors that control gene expression:

    • Activates transcription factors such as:

    • NF-κB → Inflammation and immune responses

    • Nrf2 → Antioxidant defense and detoxification

    • HIF-1α → Response to hypoxia

  • Through these factors, ROS help cells adapt to stress and metabolic changes.

4.4 Cell Proliferation and Differentiation

  • Moderate ROS levels help regulate:

    • Cell cycle progression

    • Stem cell differentiation

    • Tissue development

  • For example, ROS signalling is involved in:

    • Growth factor responses

    • Developmental signalling pathways

  • However, excessive ROS can trigger cell cycle arrest or apoptosis.

4.5 Apoptosis (Programmed Cell Death)

  • ROS play a role in regulating programmed cell death:

    • ROS can promote mitochondrial membrane permeabilization, leading to cytochrome c release and activation of caspases:

    • Controlled apoptosis is essential for:

    • Tissue homeostasis

    • Removal of damaged cells

    • Development.

4.6 Vascular Function

  • ROS contribute to the regulation of vascular tone and endothelial signalling:

    • ROS interacts with nitric oxide (NO) signalling, influencing:

    • Vasodilation

    • Blood pressure regulation

    • Endothelial cell signalling

  • Balanced ROS levels are crucial for normal cardiovascular function.

4.7 Cellular Adaptation to Stress

  • ROS act as signals that trigger adaptive stress responses, such as:

    • Antioxidant enzyme production

    • DNA repair mechanisms

    • Metabolic adjustments

  • This assists in maintaining redox homeostasis.

5. Redox Signalling Under Normal Physiological Circumstances

5.1 Core Principle: Reversible Cysteine Oxidation

  • Under normal physiological conditions:

    • Low, localized $H2O2$ is produced, diffusing short distances and being selective in oxidizing reactive cysteine residues.

  • Cysteine thiol group:

    • Reduced state: –SH

    • Oxidized to:

    • –SOH (sulfenic acid) → reversible

    • –S–S– (disulfide) → reversible

  • Important Characteristics:

    • Specificity

    • Reversibility

    • Functions like a molecular switch

  • Modifications include:

    • Enzyme activity

    • Protein–protein interactions

    • Subcellular localization

    • DNA binding.

5.2 Redox as a Signalling Switch

  • Example: Phosphatase Regulation

    • Protein tyrosine phosphatases (PTPs) contain catalytic cysteines.

    • $H2O2$ oxidizes this cysteine, leading to temporary inactivation.

  • Result:

    • Kinase activity dominates, and phosphorylation signalling is prolonged.

  • Thus, ROS:

    • Do not replace phosphorylation but modulate it—amplifying growth factor signalling.

5.3 Redox Control of Gene Transcription

  • Redox signalling affects transcription factors:

    • Direct redox regulation of transcription factors can:

    • Alter DNA-binding capacity

    • Change nuclear localization

    • Affect dimerization

  • Examples:

    • NF-κB

    • AP-1

    • p53

5.4 Indirect Regulation via Redox-Sensitive Sensor Proteins

  • KEAP1–NRF2 pathway serves as a redox sensor–effector system.

6. The KEAP1–NRF2 Pathway as a Redox Sensor–Effector System

  • Overview: Best understood as a molecular surveillance system that monitors the intracellular redox environment, adjusting gene expression accordingly rather than simply responding to oxidative damage.

  • Key Components:

    • NRF2: Nuclear factor erythroid 2-related factor 2, transcriptional activator.

    • KEAP1: Kelch-like ECH-associated protein 1, a redox-sensitive repressor.

6.1 Basal (Homeostatic) Conditions: Continuous Surveillance

  • Under normal conditions, NRF2 is continuously synthesized in the cytoplasm but does not accumulate because:

    • KEAP1 binds NRF2 immediately after translation, positioning NRF2 for ubiquitination, leading to degradation.

  • Consequences:

    • NRF2 has a short half-life (15–20 minutes).

    • Antioxidant genes are expressed at a low basal level.

    • The system remains responsive to change.

  • Role of KEAP1:

    • Contains multiple reactive cysteine residues acting as redox sensors that detect electrophiles and oxidants (like $H2O2$).

    • In the basal state, KEAP1 prevents unnecessary activation of antioxidant gene expression.

6.2 Mild Oxidative Shift: Molecular Sensing Through Cysteine Oxidation

  • When intracellular ROS levels increase (due to mitochondrial activity, inflammation, or growth factors), $H2O2$ oxidizes specific cysteine residues on KEAP1.

  • This leads to:

    • A structural conformational change in KEAP1.

    • Alters thiol (-SH) groups to forms such as sulfenic acid (-SOH) or disulfides.

    • NRF2 is no longer efficiently ubiquitinated, allowing NRF2 accumulation in the cytoplasm.

  • Key Concept: Activation of NRF2 does not turn it on; instead, it stops degradation facilitating cellular response to minor redox changes.

6.3 Nuclear Translocation and Transcriptional Activation

  • Once NRF2 accumulates, it translocates to the nucleus, where it heterodimerizes with small Maf proteins.

  • Binds to specific DNA sequences known as Antioxidant Response Elements (AREs) in target gene promoters.

  • Transcription Initiation: A coordinated gene program including:

    • Enzymes for glutathione synthesis (e.g., GCLC, GCLM)

    • Detoxifying enzymes (e.g., NQO1)

    • ROS-scavenging enzymes (e.g., SOD, GPx)

    • NADPH-generating enzymes

    • Heme oxygenase-1 (HO-1)

  • NRF2 induces a network-level metabolic reprogramming enhancing the cell’s capacity to buffer oxidative stress.

6.4 Negative Feedback and Restoration of Homeostasis

  • With increased expression of antioxidant genes, cellular reducing capacity rises:

    • Glutathione levels increase.

    • Peroxiredoxins and catalase reduce $H2O2$ levels.

    • NADPH availability improves.

  • As ROS levels revert to baseline:

    • Cysteine residues in KEAP1 return to reduced states.

    • KEAP1 resumes NRF2 ubiquitination, leading to NRF2 decline and antioxidant gene transcription returning to basal levels.

  • Conclusion: Creates a tightly regulated negative feedback loop maintaining redox homeostasis.

6.5 Why This Is a Signalling Pathway (Not Just a Stress Response)

  • The KEAP1–NRF2 pathway acts as a signalling integrator.

  • Mild and transient ROS increases during exercise, growth factor stimulation, and immune activation can activate NRF2 moderately, allowing the cell to pre-emptively enhance its oxidative stress capacity.

  • This system functions as a redox rheostat, continuously adjusting transcriptional output according to cellular oxidative tone.

7. What Is Redox Balance?

  • Definition: Redox balance (or redox homeostasis) refers to the dynamic equilibrium between oxidant production (ROS/RNS generation) and reducing capacity (antioxidant systems).

  • Important Points:

    • Redox balance does not signify zero ROS.

    • Normal physiological redox balance means:

    • ROS are at low, localized concentrations.

    • Function as signalling molecules without causing irreversible damage.

  • The body maintains a controlled oxidative tone rather than a fully reduced state.

7.1 Enzymatic Antioxidant Systems

  • Overview: Protein-based systems that actively catalyze ROS detoxification.

  • Superoxide Dismutases (SOD):

    • Function: Convert superoxide ($O2^{•-}$) into hydrogen peroxide ($H2O_2$).

    • Reaction:
      2 O2^{•-} + 2 H^+ \rightarrow H2O2 + O2

    • Isoforms:

      • SOD1 (cytosolic)

      • SOD2 (mitochondrial)

      • SOD3 (extracellular)

    • Concept: Prevents superoxide accumulation but produces $H2O2$, more stable and serves as a signalling molecule.

  • Catalase:

    • Location: Mainly peroxisomes.

    • Function: Rapidly converts hydrogen peroxide into water and oxygen.

    • Reaction:
      2 H2O2 \rightarrow 2 H2O + O2

    • Significance: Acts as a bulk detoxifier, especially at high $H2O2$ concentrations.

  • Glutathione Peroxidases (GPx):

    • Function: Reduce hydrogen peroxide or lipid peroxides using glutathione.

    • Reaction:
      H2O2 + 2 GSH \rightarrow 2 H_2O + GSSG

    • Critical for:

    • Preventing lipid peroxidation

    • Protecting membranes.

  • Peroxiredoxins (Prx):

    • Role: Vital in redox signalling.

    • React rapidly with $H2O2$.

    • Serve as antioxidants and redox signal transducers.

    • Different from catalase by regulating local $H2O2$ microdomains.

  • Thioredoxin System:

    • Components: Thioredoxin (Trx), Thioredoxin reductase, NADPH.

    • Function: Maintains protein thiols in their reduced state and reduces peroxiredoxins.

    • Critical for:

    • DNA synthesis

    • Maintaining cysteine residues in functional form.

7.2 Non-Enzymatic Antioxidants

  • These are small molecules that scavenge ROS directly.

  • Glutathione (GSH):

    • Most abundant intracellular antioxidant.

    • Functions:

    • Direct ROS scavenging.

    • Substrate for GPx.

    • Conjugation of toxins via glutathione-S-transferase.

    • Acts as the main intracellular redox buffer.

  • Vitamin C (Ascorbate):

    • Water-soluble antioxidant.

    • Functions:

    • Scavenges ROS in plasma and cytosol.

    • Regenerates vitamin E.

    • Supports iron redox balance.

  • Vitamin E (α-tocopherol):

    • Lipid-soluble antioxidant.

    • Protects:

    • Membrane lipids.

    • Prevents lipid peroxidation chain reactions.

    • Interrupts free radical propagation in membranes.

  • Uric Acid:

    • Important plasma antioxidant, scavenging peroxynitrite and hydroxyl radicals.

  • Bilirubin:

    • Formed from heme breakdown, acts as a redox buffer in circulation.

7.3 NADPH: The Central Reducing Currency

  • Importance: Central to all major antioxidant systems, supporting multiple detoxification pathways.

  • Sources of NADPH:

    • Pentose phosphate pathway

    • Malic enzyme

    • Isocitrate dehydrogenase

  • Functions:

    • Regenerate GSH from GSSG.

    • Reduce thioredoxin.

    • Maintains redox capacity.

  • Consequence: Without NADPH, antioxidant systems collapse.

8. Why ROS Can Function as Messengers

  • ROS initially seem unsuitable due to:

    • Reactivity

    • Short life span

    • Potential for damaging macromolecules.

  • Advantages of Hydrogen Peroxide ($H2O2$):

    • Relative stability compared to radicals.

    • Membrane permeability (via aquaporins).

    • Selective reactivity with certain cysteine residues.

    • Enzymatic removal, allowing reversibility.

  • Notably, hydroxyl radicals ($•OH$) are unsuitable for signalling due to their indiscriminate and damaging nature.

  • Physiological redox signalling primarily utilizes:

    • $H2O2$

    • Occasionally superoxide in microdomains.

8.1 Cysteine Chemistry — The Molecular Basis

  • Cysteine: Contains a thiol group (-SH).

    • At physiological pH, some cysteines exist in a thiolate form (-S⁻).

    • The thiolate is:

    • More nucleophilic, allowing easy binding.

    • Sensitive to oxidation.

    • Lacks a closed structure, dependent on surrounding amino acids and position within the protein.

  • Significance: Cysteine acts as an ideal redox switch due to its reactivity.

8.2 Reversible Cysteine Oxidation (Physiological Signalling)

  • When $H2O2$ reacts with a reactive cysteine thiolate:

    • Step 1: Formation of Sulfenic Acid
      –S^{-} + H2O2 \rightarrow –SOH (sulfenic acid)

    • Characteristics:

    • Unstable

    • Highly reactive

    • Reversible

  • Step 2: Possible Reversible Outcomes

    1. Form a disulfide bond:
      –SOH + –SH \rightarrow –S–S– (can be intramolecular or intermolecular).

    2. Form mixed disulfide with glutathione (S-glutathionylation):
      Protein–SH + GSH \rightarrow Protein–SSG

  • Functional Consequences:

    • Reversible cysteine oxidation can:

    • Inactivate protein tyrosine phosphatases.

    • Activate kinases.

    • Change transcription factor DNA binding.

    • Alter cytoskeletal dynamics.

  • Example: Protein tyrosine phosphatases require a reduced cysteine for catalysis. Oxidation temporarily inhibits them, enhancing phosphorylation signalling.