PRDX4 Human

What is PRDX4 and where is it localized in human cells?

PRDX4 (Peroxiredoxin 4) is a member of the peroxiredoxin family, which consists of six antioxidant isozymes (PRDX1-6). These redox family proteins are ubiquitously expressed and widely distributed across human tissues. PRDX4 is unique among the peroxiredoxins as it primarily resides in the endoplasmic reticulum (ER), containing a hydrophobic signal sequence at the N-terminus responsible for its ER localization or secretion into extracellular space .

While the other peroxiredoxins have different subcellular distributions (PRDX1, PRDX2, and PRDX6 in cytosol and nucleus; PRDX3 exclusively in mitochondria; PRDX5 in mitochondria, cytosol and peroxisomes), PRDX4 is the only ER-resident peroxiredoxin . Interestingly, recent research has identified a cytosolic isoform of PRDX4 that is transcribed from an alternative mRNA splicing uniquely in sexually matured testes .

How is PRDX4 expression distributed across human tissues?

PRDX4 shows variable expression across different human tissues. It is abundantly expressed in the heart, liver, pancreas, and muscle tissues. In contrast, PRDX4 expression is notably lower in the brain, spleen, and peripheral blood leukocytes . This tissue-specific expression pattern suggests specialized functions in different organ systems and potentially tissue-specific regulation mechanisms. Unlike other peroxiredoxins, PRDX4 is not expressed in unicellular organisms such as yeast, indicating its evolutionary development coincided with multicellularity and specialized tissue development .

What is the primary physiological function of PRDX4?

The primary physiological function of PRDX4 centers on its role in redox homeostasis within the endoplasmic reticulum. PRDX4 catalyzes the reduction of hydrogen peroxide (H₂O₂) and thereby protects the ER lumen from oxidative damage. This activity serves several critical cellular functions:

• Protein folding assistance: PRDX4 ensures the correct folding process of nascent proteins, preventing them from undergoing alternative oxidative pathways that could lead to misfolding .

• Redox balance maintenance: It couples H₂O₂ catabolism with oxidative protein folding to maintain ER redox balance .

• Signal transduction regulation: PRDX4 fine-tunes H₂O₂ concentration, which plays a critical role in cell proliferation signaling pathways .

• Protection against misfolded protein accumulation: By maintaining proper redox conditions in the ER, PRDX4 helps prevent the development and accumulation of misfolded proteins that could trigger cellular stress responses .

How does PRDX4 interact with redox shuttles and NADPH metabolism?

PRDX4 functions within a complex network of redox shuttles and NADPH metabolism. While PRDX4 itself is involved in H₂O₂ catabolism in the ER, its function is interconnected with broader cellular redox systems. The dependency of certain cancer cells on PRDX4 correlates with cellular NADPH levels, suggesting a functional relationship between PRDX4 activity and NADPH metabolism .

Several redox shuttles contribute to maintaining cellular redox balance. For instance, the pyruvate-malate shuttle enables the production of NADPH through cytosolic malic enzyme (ME1), which can then feed NADPH oxidase 4 (NOX4). Similarly, the pyruvate-isocitrate shuttle allows for the formation of NADPH that can serve as substrate for NOX4 .

PRDX4 appears to play a counterbalancing role against NOX4-generated reactive oxygen species. NOX4, which localizes to the ER membrane, has been identified as a source of ROS that creates cellular dependency on PRDX4, particularly in pancreatic cancer cells. The functional requirement for PRDX4 correlates with cellular NADPH levels across different cancer models and can be rescued by depletion of NOX4 or NADPH . This relationship highlights PRDX4's role in maintaining redox homeostasis in the face of NOX4-generated oxidative stress.

What is the "floodgate model" in relation to PRDX4 function?

The floodgate model describes a mechanism by which peroxiredoxins, including PRDX4, regulate the diffusion and signaling functions of H₂O₂. According to this model, when peroxiredoxins encounter sustained H₂O₂ flux, they can form high molecular weight (HMW) complexes. These HMW complexes effectively withdraw PRDX molecules from their catalytic cycle, preventing them from metabolizing H₂O₂ in their immediate vicinity .

This temporary inactivation of peroxiredoxins allows H₂O₂ to diffuse to more distant cellular locations, where it can oxidize and thereby activate or inactivate target proteins involved in signaling pathways. The formation of these HMW complexes thus acts as a "floodgate" that permits H₂O₂-mediated redox signaling to occur at specific locations .

The peroxiredoxins containing sulfinyls (oxidized forms) can be returned to their active state through the action of sulfiredoxin (SRX) systems. In mitochondria, this process has been linked to circadian rhythms, with SRX expression alternating with SRX degradation by LON-protease, controlled by clock genes in various tissues including the adrenal gland, brown adipose tissue, and heart .

How does PRDX4 depletion affect DNA damage and repair mechanisms?

PRDX4 depletion has significant effects on DNA integrity and repair mechanisms, particularly in cancer cells dependent on PRDX4 for survival. When PRDX4 is depleted in these cells, several consequential events occur:

• Increased ROS levels: PRDX4 knockdown results in elevated reactive oxygen species, which can directly damage DNA .

• DNA damage induction: Both single-strand breaks (SSBs) and double-strand breaks (DSBs) increase following PRDX4 depletion, as confirmed by Comet assays under alkaline and neutral conditions, respectively .

• DNA damage response activation: PRDX4 depletion leads to phosphorylation of histone H2AX at Ser139 (γH2AX), a marker of DNA double-strand breaks. This produces a focal γH2AX signal important for regulation of DNA repair .

• DNA-PKcs dependency: The DNA damage response triggered by PRDX4 depletion is primarily governed by DNA-dependent protein kinase catalytic subunit (DNA-PKcs), rather than other phosphoinositide 3-kinase-related kinase (PIKK) family members. This is evidenced by the phosphorylation of RPA32 at the DNA-PK-dependent S4/S8 residue, but not at the ATM/ATR-dependent S33 residue .

• Cell cycle effects: PRDX4 knockdown results in a higher proportion of cells in G2/M phase of the cell cycle, but only in cell lines in which PRDX4 is essential, consistent with a DNA damage-induced checkpoint activation .

These findings suggest that PRDX4 plays a crucial role in protecting cellular DNA from oxidative damage, particularly in cancer cells with elevated ROS production.

What are the recommended approaches for studying PRDX4 dependency in cancer cells?

When investigating PRDX4 dependency in cancer cells, researchers should consider the following methodological approaches:

• Genetic manipulation techniques:

  • RNA interference: Use siRNA or shRNA to deplete PRDX4. Doxycycline-inducible shRNA systems have proven effective for studying both short and long-term effects of PRDX4 depletion .

  • CRISPR-Cas9: For complete knockout studies when appropriate.

• Proliferation and survival assays:

  • Standard proliferation assays to measure growth inhibition following PRDX4 depletion

  • 3D spheroid models to assess growth in more physiologically relevant conditions. This is particularly important as cells cultured in 2D can undergo cytoskeletal rearrangements and acquire artificial polarity .

• ROS measurement:

  • Fluorescent ROS indicators to quantify changes in cellular ROS levels following PRDX4 manipulation

  • Correlation of ROS levels with PRDX4 dependency across multiple cell lines to identify sensitive and resistant populations

• DNA damage assessment:

  • Immunoblotting for γH2AX to detect double-strand breaks

  • Immunofluorescence to quantify γH2AX foci per cell

  • Comet assays under both alkaline conditions (for single-strand breaks) and neutral conditions (for double-strand breaks)

• In vivo models:

  • Establish subcutaneous or orthotopic xenografts using cells with inducible PRDX4 knockdown

  • Begin treatment after tumors are established (e.g., 200mm³) to mimic therapeutic scenarios

  • Monitor PRDX4 expression levels throughout treatment to assess potential selection of knockdown-resistant cells

• Biomarker identification:

  • Measure γH2AX as a potential functional biomarker of PRDX4 dependency

  • Assess correlation between NADPH levels and PRDX4 dependency across different cancer models

How can researchers effectively measure PRDX4 activity in experimental systems?

Measuring PRDX4 activity in experimental systems requires multiple complementary approaches:

• Protein expression analysis:

  • Western blotting for baseline PRDX4 protein levels

  • qRT-PCR for mRNA expression analysis

  • Immunohistochemistry to assess tissue localization and expression patterns

• Redox state assessment:

  • Non-reducing SDS-PAGE to visualize different redox states of PRDX4 (monomers, dimers, and high molecular weight complexes)

  • Redox Western blotting with antibodies specific to different PRDX4 oxidation states

• Peroxidase activity assays:

  • Measure H₂O₂ consumption rates in the presence of thioredoxin/thioredoxin reductase systems

  • Fluorescent or colorimetric peroxide detection assays

• Cellular ROS monitoring:

  • Use of general ROS indicators (e.g., DCFDA) or more specific probes

  • Comparison of ROS levels between wild-type and PRDX4-depleted cells under basal and stress conditions

• Functional readouts:

  • Analysis of ER stress markers (e.g., UPR-induced transcripts like XBP1s, ERdj4, and CHOP) to assess whether PRDX4 manipulation affects ER homeostasis

  • Protein folding efficiency assays to measure the impact of PRDX4 on its canonical function

• Subcellular localization:

  • Immunofluorescence or cell fractionation followed by Western blotting to confirm ER localization

  • Proximity ligation assays to identify interaction partners

• Rescue experiments:

  • Expression of wild-type versus catalytically inactive PRDX4 mutants to determine if observed phenotypes depend on peroxidase activity

  • Depletion of ROS sources (e.g., NOX4) to determine if PRDX4 dependency can be rescued

What controls should be included when studying PRDX4 in cellular redox systems?

When studying PRDX4 in cellular redox systems, the following controls should be included:

• Cell type controls:

  • Compare PRDX4-dependent and PRDX4-independent cell lines (e.g., MIA PaCa-2 and PANC-1 vs. Capan-2 and GP-3A for pancreatic cancer)

  • Include normal, non-transformed cells appropriate to the tissue being studied

• Genetic manipulation controls:

  • Non-targeting siRNA/shRNA controls

  • Multiple independent siRNA/shRNA sequences targeting PRDX4 to rule out off-target effects

  • Rescue experiments with PRDX4 cDNA resistant to the siRNA/shRNA used

• Redox state controls:

  • Positive controls for oxidative stress (e.g., H₂O₂ treatment)

  • Antioxidant treatment (e.g., N-acetylcysteine) to confirm ROS involvement

  • Treatment with specific ROS scavengers to identify critical ROS species

• Pathway inhibitor controls:

  • PIKK inhibitors (e.g., NU7441) to identify the specific DNA damage response pathways involved

  • NOX4 inhibitors or knockdown to confirm the source of ROS

  • ER stress inducers (e.g., tunicamycin, thapsigargin) as positive controls for ER stress

• Other peroxiredoxin family members:

  • Assessment of other PRDXs (especially PRDX1-3) to determine specificity of PRDX4 effects

  • Compare cytosolic (PRDX1/2) versus ER (PRDX4) versus mitochondrial (PRDX3) peroxiredoxins to determine compartment-specific effects

• Temporal controls:

  • Time-course experiments to distinguish immediate from adaptive responses

  • Inducible systems to control the timing of PRDX4 depletion

• Quantification controls:

  • Standard curves for all quantitative assays

  • Multiple technical and biological replicates

  • Statistical analysis appropriate to the experimental design

How does PRDX4 expression vary across different cancer types?

PRDX4 expression shows significant variation across cancer types, with both increased and decreased expression patterns observed depending on the cancer:

• Increased PRDX4 expression:

  • Glioblastoma: PRDX4 is increased in glioblastoma tissue compared to normal brain tissue

  • Ovarian cancer: PRDX4 expression is significantly higher in borderline than in benign epithelial ovarian tumors

  • Prostate cancer: Enhanced expression of PRDX4 is observed, which is negatively related to the TMPRSS2-ERG gene fusion status

• Decreased PRDX4 expression:

  • Acute promyelocytic leukemia (APL): Decreased PRDX4 expression negatively regulates G-CSFR mediated signaling

  • Colorectal cancer: Both irinotecan (IRI) and curcumin+irinotecan (CUR+IRI) treatments decrease PRDX4 expression in tumors implanted by LOVO cells in nude mice

• Variable expression in pancreatic cancer:

  • Approximately half of primary and established pancreatic cell lines are dependent on PRDX4 for their proliferation and survival

  • This dependency correlates with cellular NADPH levels and ROS production via NOX4

These varying expression patterns suggest context-dependent roles for PRDX4 in cancer development and progression. The dichotomous expression changes highlight the complexity of redox regulation in cancer biology and suggest that both overexpression and underexpression of PRDX4 can contribute to malignant phenotypes depending on the cancer type and cellular context.

What role does PRDX4 play in cancer cell proliferation and tumor growth?

PRDX4 exhibits diverse roles in cancer cell proliferation and tumor growth that vary by cancer type:

• Promoting cancer cell proliferation:

  • In prostate cancer, overexpression of PRDX4 remarkably improves the growth rate of cancer cell lines

  • In pancreatic cancer, approximately half of cell lines depend on PRDX4 for their proliferation and survival

  • Both established and primary pancreatic cancer cells show growth inhibition when PRDX4 is depleted, in 2D cultures, 3D spheroid models, and orthotopic xenografts

• Supporting tumor survival:

  • PRDX4 depletion induces cell death in PRDX4-dependent cancer cells, accompanied by increased levels of reactive oxygen species, DNA damage, and DNA-PKcs-governed DNA damage response

  • In mice bearing tumors from PRDX4-dependent cell lines, targeting PRDX4 increases survival

• Influencing metastatic potential:

  • PRDX4 has been identified as a secreted soluble factor produced by prostate and breast cancer cells that induces osteolastogenesis and bone destruction

  • Knockdown of PRDX4 results in less efficient induction of osteolastogenesis for both prostate and breast cancer cells

• Mediating treatment resistance:

  • In glioblastoma, PRDX4 knockdown decreases radioresistance

  • Piperlongumine treatment kills high-grade glioma cells by inactivating PRDX4 and exacerbating oxidative stress

  • PRDX4 depletion sensitizes pancreatic cancer cells and tumors to ionizing radiation

These diverse functions highlight PRDX4's context-dependent role in cancer biology and suggest potential for therapeutic targeting in specific cancer types and contexts.

How might PRDX4 be targeted therapeutically in cancer?

Based on current research, several approaches for therapeutically targeting PRDX4 in cancer can be considered:

• Direct PRDX4 inhibition:

  • RNA interference approaches (siRNA, shRNA) have demonstrated efficacy in preclinical models

  • Development of small molecule inhibitors specific to PRDX4 would be valuable, though challenging due to structural similarities with other peroxiredoxins

  • Targeting the unique N-terminal signal sequence of PRDX4 might provide specificity

• Synthetic lethality approaches:

  • Targeting PRDX4 in combination with radiation therapy, as PRDX4 depletion sensitizes cells to ionizing radiation

  • Combining PRDX4 inhibition with DNA damaging agents, given that PRDX4 depletion induces DNA damage and activates DNA damage response pathways

  • Dual targeting of PRDX4 and DNA-PK, as DNA-PK activation appears protective following PRDX4 depletion

• Targeting upstream regulators:

  • Inhibiting NOX4, which generates the ROS that creates dependency on PRDX4

  • Modulating NADPH levels, as PRDX4 dependency correlates with cellular NADPH levels

• Cancer type-specific approaches:

  • For prostate and breast cancers: Target secreted PRDX4 to prevent bone metastasis and osteolastogenesis

  • For glioblastoma: Combine PRDX4 inhibition with radiotherapy to overcome radioresistance

  • For pancreatic cancer: Identify biomarkers (e.g., NOX4 expression, NADPH levels) to select patients likely to respond to PRDX4-targeted therapy

• Biomarker-guided therapy:

  • Use γH2AX as a functional biomarker to identify tumors likely to respond to PRDX4 inhibition

  • Measure ROS levels as predictive biomarkers for PRDX4 dependency

• Targeting the secreted form:

  • Develop antibodies against extracellular PRDX4 to prevent its role in bone metastasis

  • Block PRDX4 from functioning as a secreted soluble factor in the tumor microenvironment

These approaches require further validation in preclinical models before advancing to clinical trials, but they represent promising strategies for exploiting PRDX4 dependency in cancer therapy.

What are the main technical challenges in studying PRDX4 function?

Researchers face several technical challenges when studying PRDX4 function:

• Distinguishing PRDX4 from other peroxiredoxins:

  • High sequence homology among peroxiredoxin family members can complicate specific detection

  • Need for highly specific antibodies that do not cross-react with other PRDX proteins

  • Challenge of generating PRDX4-specific inhibitors due to structural similarities

• Compartment-specific redox measurements:

  • Accurately measuring ROS specifically within the ER lumen where PRDX4 primarily functions

  • Distinguishing between cytosolic and ER-localized PRDX4 isoforms

  • Monitoring real-time changes in PRDX4 oxidation state in living cells

• Temporal dynamics of redox signaling:

  • Capturing rapid redox reactions and signaling events

  • Distinguishing between immediate PRDX4 functions and adaptive responses

  • Monitoring the transition between different PRDX4 oxidation states

• In vivo models and translation:

  • Development of appropriate animal models to study PRDX4 function

  • Potential differences in redox biology between humans and model organisms

  • Translating findings from cell culture to in vivo settings

• Context dependency:

  • Understanding why certain cell lines are dependent on PRDX4 while others are not

  • Elucidating the interplay between PRDX4 and other redox systems

  • Accounting for microenvironmental factors that influence PRDX4 function

• Long-term inhibition challenges:

  • Selection pressure leading to resistance mechanisms when targeting PRDX4

  • Transgene silencing or other adaptation mechanisms affecting long-term response to PRDX4 depletion

  • Need for inducible systems to study acute versus chronic PRDX4 inhibition

How do contradictory findings about PRDX4 in different cancer types get reconciled?

Contradictory findings regarding PRDX4 in different cancer types can be reconciled through several conceptual frameworks:

• Context-dependent role based on redox environment:

  • Cancer cells exist on a spectrum of redox states, from highly oxidized to more reduced

  • PRDX4 may be protective in highly oxidized environments but dispensable in more reduced settings

  • The correlation between PRDX4 dependency and cellular NADPH levels supports this model

• Cancer-specific metabolic adaptations:

  • Different cancers adopt distinct metabolic profiles that may create varying degrees of dependence on redox systems

  • Cancers with high ER stress or secretory burden may be more dependent on PRDX4 function

  • The source and quantity of ROS production (e.g., NOX4 activity) likely influences PRDX4 dependency

• Integration with complementary redox systems:

  • Redundancy in redox systems may explain why some cancers are PRDX4-independent

  • The relative expression of other peroxiredoxins or antioxidant enzymes may determine PRDX4 dependency

  • The capacity of alternative H₂O₂ scavenging systems may compensate for PRDX4 depletion in some contexts

• Dual roles in cancer progression:

  • PRDX4 may play different roles at different stages of cancer development

  • Initial protective functions may transition to pro-tumorigenic roles during cancer evolution

  • The secreted versus ER-resident forms of PRDX4 may have distinct functions

• Methodological considerations:

  • Different experimental systems (cell lines, primary cells, animal models, human samples)

  • Varying methods of PRDX4 manipulation (transient vs. stable, partial vs. complete depletion)

  • Different readouts and endpoints measured across studies

• Integration with genetic background:

  • Specific genetic alterations in different cancers may determine PRDX4 dependency

  • For example, in prostate cancer, PRDX4 expression is negatively related to the TMPRSS2-ERG gene fusion status

Reconciling these contradictions requires comprehensive characterization of PRDX4 function across multiple cancer types, with careful attention to cellular context, genetic background, and metabolic state.

What are promising future directions for PRDX4 research?

Several promising future directions for PRDX4 research emerge from current findings:

• Single-cell resolution studies:

  • Investigating PRDX4 function and dependency at single-cell level to capture heterogeneity

  • Correlating PRDX4 activity with specific cellular states and phenotypes

  • Mapping PRDX4 oxidation dynamics in real-time using advanced redox biosensors

• Multi-omics integration:

  • Combining proteomics, metabolomics, and transcriptomics to build comprehensive models of PRDX4 function

  • Identifying metabolic signatures that predict PRDX4 dependency

  • Mapping the PRDX4 interactome under different cellular conditions

• Translational applications:

  • Developing PRDX4 as a biomarker for patient stratification in cancer therapy

  • Creating PRDX4-targeting therapeutic approaches for PRDX4-dependent cancers

  • Exploring combination therapies that exploit PRDX4 dependency

• Mechanistic studies:

  • Further characterizing the DNA damage induced by PRDX4 depletion

  • Investigating the relationship between PRDX4 and circadian rhythms

  • Understanding how PRDX4 coordinates with unfolded protein response pathways

• Systems biology approaches:

  • Mathematical modeling of redox shuttles and their relationship with PRDX4

  • Network analysis of PRDX4 interactions within broader redox systems

  • Prediction of synthetic lethal interactions with PRDX4

• Therapeutic development:

  • Design of specific PRDX4 inhibitors

  • Development of strategies to target secreted versus ER-resident PRDX4

  • Exploration of immunotherapeutic approaches targeting PRDX4 in the tumor microenvironment

• Role in non-cancer diseases:

  • Investigating PRDX4 in inflammatory diseases, diabetes, and neurodegenerative disorders

  • Exploring PRDX4 as a biomarker for various diseases beyond cancer

  • Understanding the role of PRDX4 in normal physiology and aging

These directions will advance our understanding of PRDX4 biology and potentially lead to new diagnostic and therapeutic approaches for cancer and other diseases.