PRDX3 Human

What is PRDX3 and what is its primary cellular localization?

PRDX3 (Thioredoxin-dependent peroxide reductase, mitochondrial) is a member of the peroxiredoxin family of antioxidant enzymes encoded by the PRDX3 gene in humans . It is localized exclusively in the mitochondrial matrix, where it functions as the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide generated as a byproduct of cellular respiration from the mitochondrial electron transport chain . This mitochondrial localization can be confirmed through cell fractionation and confocal microscopy analyses, as demonstrated in studies with HEK293T cells .

How does PRDX3 function in maintaining cellular redox homeostasis?

PRDX3 plays a crucial role in maintaining cellular redox homeostasis by scavenging reactive oxygen species (ROS), particularly hydrogen peroxide, in the mitochondria . As mitochondria are major sites of ROS production through the respiratory chain, PRDX3 provides a primary defense mechanism against oxidative damage . When mitochondrial ROS levels increase beyond a cytotoxic threshold, they can activate stress signaling pathways and cause cellular damage . PRDX3 prevents this by eliminating excess hydrogen peroxide, thus protecting mitochondrial integrity and function. In PRDX3-knockdown THP-1 cells, significantly higher mitochondrial ROS levels have been observed compared to control cells, demonstrating PRDX3's critical role in ROS regulation .

What transcriptional regulation controls PRDX3 expression?

PRDX3 is a direct target gene of the c-Myc transcription factor . Expression of PRDX3 is induced by the mycER system and is reduced in c-myc−/− cells . Chromatin immunoprecipitation analysis has revealed that Myc binds preferentially to a 930-bp region surrounding exon 1 of the PRDX3 gene . This regulatory relationship is significant as c-Myc is a key transcription factor implicated in the control of various cellular processes including cell growth, cell-cycle progression, and apoptosis, and its deregulated expression is common in many human tumors .

How does PRDX3 contribute to mitochondrial integrity and function?

PRDX3 is essential for maintaining mitochondrial mass and membrane potential in transformed rat and human cells . Research using mitochondria-specific fluorescent probes has demonstrated that PRDX3 maintains normal mitochondrial function, which is critical for cellular energy production and viability . When PRDX3 is knocked down, cells exhibit mitochondrial dysfunction characterized by decreased mitochondrial membrane potential and altered mitochondrial mass . Moreover, in studies of intrahepatic cholestasis of pregnancy (ICP), toxic levels of bile acids and PRDX3 knockdown induced oxidative stress and mitochondrial dysfunction in trophoblast cells .

What is the relationship between PRDX3 and cellular senescence?

Silencing of PRDX3 in the trophoblast cell line HTR8/SVneo has been shown to induce growth arrest and cellular senescence through activation of p38-mitogen-activated protein kinase (MAPK) and induction of p21 WAF1/CIP and p16 INK4A . This enhanced cellular senescence, as determined by senescence-associated beta-galactosidase staining, can be significantly attenuated by the p38-MAPK inhibitor SB203580 . These findings suggest that PRDX3 plays a protective role against cellular senescence by preventing excessive oxidative stress that can trigger senescence-associated signaling pathways.

What role does PRDX3 play in ferroptosis?

Hyperoxidized PRDX3 has been identified as a specific marker for ferroptosis both in vitro and in vivo . During ferroptosis, a regulated cell death pathway driven by accumulation of phospholipid peroxides, mitochondrial lipid peroxides trigger PRDX3 hyperoxidation . This posttranslational modification converts a cysteine thiol to sulfinic or sulfonic acid . Interestingly, once hyperoxidized, PRDX3 translocates from mitochondria to plasma membranes where it inhibits cystine uptake, thereby causing ferroptosis . Studies in various cell lines including SV589, HT1080, A549, Huh7, and HT29 cells have shown that ferroptosis inducers such as erastin and RSL3 trigger hyperoxidation of PRDX3 in a concentration-dependent manner .

What CRISPR-based approaches can be used to target PRDX3 in human cells?

For CRISPR-based targeting of PRDX3, researchers can utilize guide RNA sequences designed by Feng Zhang's laboratory at the Broad Institute . These sequences uniquely target the PRDX3 gene within the human genome with minimal risk of off-target Cas9 binding . When implementing CRISPR-Cas9 for PRDX3 knockout studies, it's recommended to order at least two gRNA constructs to increase the chance of successful gene targeting . Researchers should verify the gRNA sequences against their target gene sequence before ordering, especially when targeting specific splice variants or exons . These CRISPR tools enable precise manipulation of PRDX3 expression for functional studies.

How can researchers detect and measure PRDX3 hyperoxidation in experimental systems?

PRDX3 hyperoxidation can be detected using antibodies specific to hyperoxidized peroxiredoxins (anti-PrxSO2/3) in western blotting assays . To confirm that the hyperoxidized peroxiredoxin is specifically PRDX3, researchers can perform immunoprecipitation with anti-PRDX3 antibodies followed by western blotting with anti-PrxSO2/3 antibodies . Additionally, comparing wildtype cells with PRDX3 knockout cells can verify the specificity of hyperoxidized PRDX3 signals, as the signal should be absent in knockout cells . For localization studies, immunofluorescent microscopy can distinguish between normal PRDX3 (mitochondrial) and hyperoxidized PRDX3 (plasma membrane) .

What methods are effective for studying PRDX3's role in mitochondrial ROS regulation?

To study PRDX3's role in mitochondrial ROS regulation, researchers can employ several complementary approaches:

• Genetic manipulation: PRDX3 knockdown using siRNA or CRISPR-based knockout

• ROS measurement: Mitochondria-specific fluorescent probes for detecting mitochondrial ROS levels

• Flow cytometry analysis: For quantitative assessment of mitochondrial ROS in control versus PRDX3-deficient cells

• Cellular fractionation: To confirm mitochondrial localization of PRDX3 and to study its translocation during oxidative stress conditions

• Bacterial infection assays: For example, using Salmonella enterica serovar Typhimurium to assess the impact of PRDX3 on bactericidal activity through mROS regulation

Research has shown that PRDX3 knockdown THP-1 cells exhibit significantly higher mitochondrial ROS levels than control cells, and these levels increase markedly upon lipopolysaccharide (LPS) stimulation .

How is PRDX3 implicated in liver disorders?

PRDX3 has been implicated in multiple liver disorders through distinct mechanisms:

• Hepatocellular carcinoma: Serum PRDX3 has been demonstrated to be a valuable biomarker for the diagnosis and assessment of hepatocellular carcinoma .

• Alcoholic and nonalcoholic fatty liver diseases: Hyperoxidized PRDX3 serves as a marker for ferroptosis in mouse models of both conditions, suggesting that ferroptosis is responsible for hepatocyte death in these prevalent chronic liver disorders .

• Therapeutic potential: The identification of ferroptosis as a mechanism of hepatocyte death opens the possibility to treat these liver diseases with drugs that inhibit ferroptosis .

The dual role of PRDX3 as both a biomarker and a mechanistic player in liver pathology makes it a valuable target for both diagnostic and therapeutic development in liver diseases.

What is the relationship between PRDX3 and intrahepatic cholestasis of pregnancy (ICP)?

PRDX3 is significantly downregulated in ICP placentas as well as in bile acid-treated trophoblast cells and villous explants in vitro . This downregulation contributes to the pathogenesis and progression of ICP through several mechanisms:

• Oxidative stress induction: Toxic levels of bile acids and PRDX3 knockdown induce oxidative stress and mitochondrial dysfunction in trophoblast cells .

• Cellular senescence: Decreased PRDX3 leads to enhanced cellular senescence in trophoblasts via activation of p38-MAPK and induction of senescence markers p21 WAF1/CIP and p16 INK4A .

• Placental dysfunction: The resulting trophoblast dysfunction impairs placental function, contributing to the adverse fetal outcomes associated with ICP .

These findings suggest that PRDX3 plays a protective role in maintaining normal trophoblast function during pregnancy, and its downregulation by excessive bile acids contributes to the pathophysiology of ICP.

How does PRDX3 contribute to cancer cell survival and resistance to therapy?

PRDX3 has been shown to contribute to cancer cell survival and therapy resistance through multiple mechanisms:

• Protection against oxidative stress: PRDX3 is overexpressed in prostate cancer and promotes cancer cell survival by defending cells against the damages incurred by oxidative stress .

• Chemotherapy resistance: Peroxiredoxin proteins, including PRDX3, protect MCF-7 breast cancer cells against doxorubicin-mediated toxicity .

• Mitochondrial function maintenance: As a c-Myc target gene, PRDX3 is required for Myc-mediated proliferation and transformation in cancer cells, maintaining mitochondrial function necessary for rapid cancer cell growth .

• Cell death regulation: PRDX3 can influence ferroptosis, a form of regulated cell death, potentially affecting the sensitivity of cancer cells to therapies that induce this pathway .

Understanding PRDX3's role in cancer may provide opportunities for developing targeted therapies that disrupt its protective functions, potentially sensitizing cancer cells to existing treatments.

How does hyperoxidized PRDX3 translocation impact cellular signaling pathways?

Hyperoxidation of PRDX3 during ferroptosis leads to its translocation from mitochondria to plasma membranes . This translocation has significant functional consequences:

• Inhibition of cystine uptake: At the plasma membrane, hyperoxidized PRDX3 inhibits cystine uptake, which further promotes ferroptosis by limiting the cell's ability to synthesize glutathione, a key antioxidant .

• Altered cellular signaling: The translocation represents a critical link between mitochondrial oxidative stress and plasma membrane function, suggesting an integrated cellular response to oxidative damage.

• Amplification mechanism: This process creates a positive feedback loop that amplifies ferroptotic cell death, as decreased cystine uptake leads to further oxidative stress.

What is the interplay between PRDX3 and other peroxiredoxin family members in cellular redox homeostasis?

While PRDX3 is specifically localized to mitochondria, other peroxiredoxin family members (PRDX1-6) are distributed across different cellular compartments and contribute collectively to redox homeostasis:

This interplay between PRDX family members highlights the complexity of cellular redox regulation and suggests that targeting specific PRDXs might allow for compartment-specific modulation of redox states in therapeutic applications.

How do post-translational modifications of PRDX3 beyond hyperoxidation affect its function?

While hyperoxidation of PRDX3 has been well-studied in the context of ferroptosis, other post-translational modifications may also significantly impact PRDX3 function:

• Reversible oxidation: The catalytic cycle of PRDX3 involves reversible oxidation of its peroxidatic cysteine to sulfenic acid, which is then resolved by reaction with a resolving cysteine from another PRDX3 subunit, forming a disulfide bond .

• Sulfinylation vs. sulfonylation: The hyperoxidation of PRDX3 can result in either sulfinic (–SO2H) or sulfonic (–SO3H) acid modifications of critical cysteine residues, with potentially different functional outcomes .

• Reduction by sulfiredoxin: Unlike sulfonylation, sulfinylation can be reversed by sulfiredoxin, suggesting a potential regulatory mechanism for PRDX3 activity under oxidative stress conditions.

Understanding the complete spectrum of PRDX3 post-translational modifications and their functional consequences represents an important area for future research, potentially revealing new regulatory mechanisms and therapeutic targets.

What experimental contradictions exist in PRDX3 research and how might they be resolved?

Several experimental contradictions or uncertainties exist in the PRDX3 research field:

• Cell-type specific effects: PRDX3 knockout renders cells more resistant to erastin-induced ferroptosis , yet PRDX3 is generally considered protective against oxidative stress . This apparent contradiction may reflect cell-type specific effects or context-dependent functions of PRDX3.

• Dual role in cell death: PRDX3 is required for Myc-mediated apoptosis after glucose withdrawal , yet it also protects against oxidative stress-induced cell death in other contexts . Resolving this contradiction requires careful examination of the specific cellular context and death pathways involved.

• Hyperoxidation consequence vs. cause: Whether PRDX3 hyperoxidation is a cause or consequence of ferroptosis remains debated. Research comparing the timing of PRDX3 hyperoxidation with other ferroptotic events could help resolve this question.

These contradictions highlight the complexity of PRDX3 biology and suggest that integrated approaches examining multiple aspects of PRDX3 function simultaneously in well-defined experimental systems are needed to resolve these discrepancies.

What novel therapeutic strategies might target PRDX3 in disease contexts?

Based on current understanding of PRDX3 biology, several novel therapeutic strategies could be explored:

• Ferroptosis modulation: For conditions where ferroptosis contributes to pathology (such as alcoholic and nonalcoholic fatty liver diseases), preventing PRDX3 hyperoxidation or its translocation to plasma membranes might inhibit ferroptotic cell death .

• Cancer therapy sensitization: In cancers where PRDX3 is overexpressed and contributes to therapy resistance, inhibiting PRDX3 function might sensitize cells to conventional treatments or to ferroptosis inducers .

• Mitochondrial protection: For conditions characterized by mitochondrial dysfunction and oxidative stress (like ICP), enhancing PRDX3 expression or activity might protect mitochondrial function and prevent cellular senescence .

• Bactericidal enhancement: Considering PRDX3's role in regulating mitochondrial ROS involved in bactericidal activity, temporary inhibition of PRDX3 might enhance immune cell function against bacterial infections .

Each of these strategies would require careful consideration of the tissue-specific and context-dependent functions of PRDX3 to avoid unintended consequences.

How might emerging technologies advance PRDX3 research?

Several emerging technologies hold promise for advancing PRDX3 research:

• Single-cell redox imaging: Technologies that allow real-time visualization of redox changes and PRDX3 activity at the single-cell level could provide unprecedented insights into the dynamics of PRDX3 function.

• Organoid models: Patient-derived organoids could enable the study of PRDX3 in complex tissue environments that more accurately reflect human disease conditions.

• PROTAC technology: Proteolysis targeting chimeras (PROTACs) could enable more selective and rapid degradation of PRDX3 than genetic knockout approaches, allowing better temporal control in functional studies.

• Mitochondria-targeted therapies: Advances in mitochondria-targeted drug delivery systems could enable specific modulation of PRDX3 activity within its native mitochondrial environment.

• Machine learning approaches: Computational methods could help integrate diverse datasets on PRDX3 function, interaction partners, and disease associations to identify new research directions and therapeutic opportunities.