Recombinant Neurospora crassa Oxidation resistance protein 1 (oxr-1)

What is the function of Oxidation resistance protein 1 (oxr-1) in Neurospora crassa?

Oxidation resistance protein 1 (oxr-1) in Neurospora crassa, similar to its homologs in other organisms, functions primarily as a protective protein against oxidative stress. Based on studies in related systems, oxr-1 likely controls the sensitivity of fungal cells to reactive oxygen species (ROS) and plays a vital role in the oxidative stress response pathway. In mammals, Oxr1 has been shown to control neuronal cell sensitivity to oxidative stress, with mice lacking Oxr1 displaying cerebellar neurodegeneration . Similarly, in the fungal pathogen Aspergillus fumigatus, OxrA localizes to mitochondria and is essential for oxidative stress resistance . The conservation of this protein across eukaryotes suggests a fundamental role in ROS detoxification mechanisms.

How is recombinant N. crassa oxr-1 typically expressed and purified for research?

Recombinant N. crassa oxr-1 can be expressed using several heterologous expression systems. Based on established protocols for N. crassa proteins, the following methodology is recommended:

Expression Systems:

• Pichia pastoris: Offers high expression levels with proper protein folding and post-translational modifications. The protocol involves cloning the oxr-1 gene into an expression vector under the control of the AOX1 methanol-inducible promoter, similar to methods used for N. crassa cellobiose dehydrogenase .

• E. coli: For faster production, though may require optimization for fungal protein folding.

Purification Protocol:

• Clone the N. crassa oxr-1 coding sequence (without native signal sequence) fused with a His6-tag

• Express in the chosen system (P. pastoris recommended for fungal proteins)

• Harvest cells after induction (typically 2-3 days for P. pastoris)

• Lyse cells and clarify lysate by centrifugation

• Purify using Ni-NTA affinity chromatography under non-denaturing conditions

• Assess purity by SDS-PAGE

• Store in Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for long-term storage

What are the optimal storage conditions for maintaining recombinant N. crassa oxr-1 activity?

Based on general guidelines for recombinant fungal proteins similar to oxr-1, the following storage conditions are recommended:

Repeated freezing and thawing should be avoided as it may lead to protein denaturation and loss of activity . It is advisable to prepare small working aliquots of the purified protein to minimize freeze-thaw cycles.

How can I evaluate the oxidative stress response function of recombinant N. crassa oxr-1 in vitro?

To evaluate the oxidative stress response function of recombinant N. crassa oxr-1 in vitro, the following assays can be implemented:

Direct H₂O₂ Scavenging Assay:

• Purify recombinant oxr-1 protein (concentration range: 0.1-10 μM)

• Use the Amplex Red assay to quantify decreasing H₂O₂ concentration in the presence of increasing concentrations of oxr-1

• Include controls without horseradish peroxidase (HRP) to confirm that oxr-1 is not compensating for HRP activity

• Calculate the rate constant for oxr-1 oxidation by H₂O₂

Based on mammalian Oxr1 studies, you should expect a rate constant of approximately 0.8 M⁻¹·s⁻¹, which is lower than dedicated antioxidant enzymes but sufficient to indicate reactivity with ROS .

Cysteine Oxidation Analysis:

• Incubate recombinant oxr-1 with varying concentrations of H₂O₂ (0.1-10 mM)

• Analyze oxidation state of cysteine residues using mass spectrometry

• Identify specific cysteine residues susceptible to oxidation (look for conserved cysteine residues in the TLDc domain)

What approaches can be used to study the function of oxr-1 in N. crassa through genetic manipulation?

For comprehensive functional analysis of oxr-1 in N. crassa, a multi-faceted genetic approach is recommended:

Knockout Strategy:

• Generate oxr-1 deletion cassette using homologous recombination

  • Amplify ~1kb flanking regions of the oxr-1 gene

  • Use N. crassa pyr-4 or another selectable marker

  • Transform the deletion cassette into wild-type N. crassa

• Verify transformants by diagnostic PCR

• Test phenotypes under oxidative stress conditions (H₂O₂, menadione, paraquat)

Complementation Analysis:

• Clone the wild-type oxr-1 gene with native promoter into a vector with a different selectable marker

• Transform the complementation construct into the oxr-1 knockout strain

• Assess whether wild-type phenotype is restored under oxidative stress

Overexpression Studies:

• Place the oxr-1 gene under the control of a strong promoter (e.g., ccg-1)

• Transform into wild-type N. crassa

• Evaluate enhanced resistance to oxidative stress agents

Based on studies with Aspergillus fumigatus OxrA, you should expect that deletion of oxr-1 will increase sensitivity to H₂O₂, while overexpression may enhance resistance .

How does N. crassa oxr-1 compare structurally and functionally to its homologs in other species?

N. crassa oxr-1 belongs to the evolutionarily conserved oxidation resistance protein family found across eukaryotes. Comparative analysis reveals:

Structural Conservation:
The most conserved region of oxr-1 is the C-terminal TLDc domain, which is present in all eukaryotic Oxr1 homologs. This domain was originally predicted to have catalytic activity . The conservation suggests a fundamental role in oxidative stress resistance.

Functional Comparison Table:

The high degree of functional conservation suggests that N. crassa oxr-1 likely plays a similar role in oxidative stress resistance as its homologs in other species, potentially through regulation of antioxidant enzyme activity or direct interaction with ROS.

What is the relationship between oxr-1 and cellular antioxidant systems in N. crassa?

Based on studies of Oxr1 homologs in other systems, N. crassa oxr-1 likely functions as a regulator of antioxidant systems rather than as a primary antioxidant enzyme. The proposed relationships include:

Regulation of Catalase Activity:
In A. fumigatus, OxrA regulates catalase function, and overexpression of catalase can rescue phenotypes associated with OxrA deficiency . In N. crassa, oxr-1 likely interacts with one or more of the catalase genes to modulate H₂O₂ detoxification.

Potential Interactions with Other Antioxidant Systems:

• Superoxide dismutases (SODs) - May regulate SOD expression or activity

• Glutathione peroxidases (GPXs) - In Anopheles gambiae, Oxr1 regulates GPX levels

• Peroxiredoxins - May coordinate with these thiol-specific antioxidants

To investigate these relationships experimentally:

• Measure catalase, SOD, and GPX activities in wild-type vs. oxr-1 deletion strains

• Perform qRT-PCR to assess transcriptional changes in antioxidant genes

• Use co-immunoprecipitation to identify direct protein-protein interactions

• Test genetic interactions through double knockout studies (oxr-1 with various antioxidant genes)

How does recombinant N. crassa oxr-1 respond to different types of oxidative stressors in vitro?

Understanding how recombinant oxr-1 responds to different oxidative stressors provides insights into its protective mechanisms. The following experimental approach is recommended:

Comparative Stress Response Assay:

• Purify recombinant oxr-1 protein to >85% homogeneity

• Expose the protein to equimolar concentrations of different oxidants:

  • H₂O₂ (hydrogen peroxide) - direct oxidant

  • Menadione - superoxide generator

  • t-BOOH (tert-butyl hydroperoxide) - organic peroxide

  • AAPH (2,2'-Azobis(2-amidinopropane) dihydrochloride) - peroxyl radical generator

  • SIN-1 (3-morpholinosydnonimine) - peroxynitrite generator

• Analyze protein modifications using:

  • Mass spectrometry to identify oxidized residues

  • Circular dichroism to assess structural changes

  • Functional assays to measure remaining activity

Expected Results:
Based on mammalian Oxr1 studies, you would expect preferential oxidation of specific cysteine residues in the TLDc domain when exposed to H₂O₂ . The protein may show different patterns of modification depending on the oxidant, providing insights into its specificity and mechanism of action.

What are the potential applications of recombinant N. crassa oxr-1 in studying fungal stress responses?

Recombinant N. crassa oxr-1 offers versatile applications for investigating fungal stress responses:

Research Applications:

• Biomarker for Oxidative Stress:

  • Develop antibodies against recombinant oxr-1

  • Monitor oxr-1 expression/modification as a marker of oxidative stress in various fungal systems

• Model System for Studying Conserved Stress Responses:

  • Compare oxr-1 function across fungal species

  • Investigate evolutionary conservation of oxidative stress defense mechanisms

• Tool for Screening Antifungal Compounds:

  • Use oxr-1-deficient N. crassa as a sensitized background for drug screening

  • Identify compounds that specifically target oxidative stress response pathways

• Investigation of ROS Signaling Networks:

  • Use recombinant oxr-1 to identify interaction partners

  • Map the signaling networks connecting oxidative stress detection to cellular responses

When implementing these applications, researchers should consider using N. crassa as a model organism due to its advantages of robust and quick growth, ease of genetic manipulation, and availability of molecular tools and mutants .

How can site-directed mutagenesis of recombinant N. crassa oxr-1 inform structure-function relationships?

Site-directed mutagenesis of conserved residues in recombinant N. crassa oxr-1 can provide critical insights into its mechanism of action:

Key Target Residues:

• Conserved cysteines in the TLDc domain - potential sites for oxidation

• Residues conserved between fungal and mammalian Oxr1 proteins

• Alanine residue in the TLDc domain that is mutated in human familial infantile myoclonic epilepsy (FIME) when present in TBC1D24

Experimental Approach:

• Generate a panel of point mutants focusing on:

  • Cysteine to serine mutations (to prevent oxidation)

  • Mutations corresponding to known human disease variants

  • Alanine scanning of conserved residues

• Characterize each mutant for:

  • H₂O₂ reactivity using Amplex Red assay

  • Structural integrity using circular dichroism

  • Protein stability under oxidative conditions

  • Ability to complement oxr-1 deletion in vivo

Expected Outcomes:
Mutation of key cysteine residues should reduce the reactivity with H₂O₂ if direct oxidation is a primary mechanism. Based on mammalian Oxr1 studies, cysteines equivalent to Cys753 and Cys704 in human Oxr1 would be of particular interest, as these residues have different accessibility to peroxide .