Recombinant Drosophila erecta Serine protease HTRA2, mitochondrial (HtrA2)
Description
Introduction to Recombinant Drosophila erecta Serine Protease HTRA2, Mitochondrial (HtrA2)
Recombinant Drosophila erecta Serine protease HTRA2, mitochondrial (HtrA2), is a recombinant protein derived from the fruit fly species Drosophila erecta. This protein is part of the HtrA family of serine proteases, which play crucial roles in maintaining mitochondrial integrity and regulating cellular stress responses. The HtrA2 protein is particularly notable for its involvement in mitochondrial function and its potential role in neurodegenerative diseases.
Production and Characteristics
The recombinant HtrA2 protein from Drosophila erecta is typically produced in either yeast or Escherichia coli (E. coli) expression systems. The choice of host organism can affect the protein's yield, purity, and post-translational modifications. For instance, yeast systems are often used for proteins requiring complex modifications, while E. coli is favored for high-yield production of simpler proteins.
| Production Characteristics | Description |
|---|---|
| Source | Yeast or E. coli |
| Form | Partial protein, often lyophilized powder |
| Purity | High purity, typically >90% as determined by SDS-PAGE |
| Storage | Store at -20°C/-80°C to maintain stability |
Biological Function
HtrA2 proteins are known for their serine protease activity, which is essential for maintaining mitochondrial homeostasis. They are involved in the degradation of misfolded or damaged proteins within mitochondria, thus preventing oxidative stress and promoting cellular survival. In Drosophila, HtrA2 has been implicated in pathways related to stress resistance and male fertility, similar to its mammalian counterparts .
Research Findings
Research on HtrA2 in Drosophila species has highlighted its role in mitochondrial integrity and its interaction with other genes involved in neurodegenerative diseases, such as PINK1 and parkin. Studies have shown that while HtrA2 is not crucial for apoptosis, it acts in a pathway parallel to Parkin and downstream of PINK1, suggesting a complex interplay in maintaining mitochondrial function .
| Species | Function | Interaction |
|---|---|---|
| Drosophila melanogaster | Maintains mitochondrial integrity, involved in stress resistance and male fertility | Acts downstream of PINK1, parallel to Parkin |
| Drosophila erecta | Presumed similar roles due to conserved HtrA2 function across species | Likely interacts with similar pathways |
Applications in Research
Recombinant HtrA2 proteins are valuable tools for studying mitochondrial biology and the mechanisms underlying neurodegenerative diseases. They can be used in biochemical assays to investigate protein-protein interactions, proteolytic activity, and the regulation of cellular stress responses.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for fulfillment according to your requirements.
Note: All proteins are shipped with standard blue ice packs unless dry ice shipping is requested in advance. Additional fees apply for dry ice shipping.
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Target Background
KEGG: der:Dere_GG21285
Q&A
FAQs for Researchers: Recombinant Drosophila erecta Serine Protease HTRA2, Mitochondrial (HtrA2)
What is the primary biological function of HTRA2 in Drosophila erecta?
Key Functional Domains:
Which expression systems are optimal for producing recombinant Drosophila erecta HTRA2?
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Bacterial Expression: Use codon-optimized constructs with N-terminal His-tags for affinity purification .
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Mitochondrial Processing: Co-express with chaperones (e.g., GroEL/ES) to ensure proper folding of the protease domain .
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Activity Validation: Post-purification, confirm proteolytic competence using fluorogenic substrates (e.g., H2-Opt) or casein degradation assays .
How can researchers confirm the proteolytic activity of recombinant HTRA2 in vitro?
Methodology:
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Fluorogenic Assays: Use H2-Opt substrate (Innovagen) to measure cleavage kinetics. Activity is quantified via fluorescence increase (Ex/Em: 380/460 nm) .
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Casein Zymography: Resolve HTRA2 by non-reducing SDS-PAGE embedded with β-casein. Proteolysis manifests as clear bands post-Coomassie staining .
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Inhibitor Controls: Include serine protease inhibitors (e.g., PMSF, UCF-101) to confirm activity specificity .
Example Data:
| Condition | H2-Opt Cleavage (RFU/min) | Casein Degradation (%) |
|---|---|---|
| WT HTRA2 | 450 ± 30 | 85 ± 5 |
| S276C Mutant | 15 ± 5 | 10 ± 3 |
| + UCF-101 (30µM) | 20 ± 8 | 12 ± 4 |
What experimental approaches resolve contradictions in HTRA2's pro-apoptotic vs. mitochondrial protective roles?
Contradictory findings arise from model-specific contexts:
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Pro-apoptotic Role: RNAi knockdown in Drosophila larvae reduces UV-induced apoptosis, while overexpression cleaves DIAP1, triggering caspase activation .
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Protective Role: HtrA2 null mutants exhibit mitochondrial fragmentation and sensitivity to rotenone, linking it to PINK1/Parkin-mediated mitophagy .
Resolution Strategies:
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Tissue-Specific Knockdown: Use TH-Gal4 (dopaminergic neurons) vs. Act5C-Gal4 (ubiquitous) drivers to assess context-dependent phenotypes .
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Stress Gradients: Titrate apoptotic stimuli (e.g., H₂O₂) to separate low-level homeostatic vs. high-level apoptotic functions .
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Genetic Epistasis: Test HtrA2 mutants with PINK1 or parkin deletions to map pathway hierarchy .
How does HTRA2 interact with Parkinson’s disease-associated proteins like PINK1/Parkin in Drosophila models?
HTRA2 acts downstream of PINK1 but parallel to Parkin in mitochondrial quality control:
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Phosphorylation Dependency: PINK1 phosphorylates HTRA2 at Ser142/Ser212, enhancing its protease activity .
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Phenotypic Rescue: Co-expressing Buffy (anti-apoptotic Bcl-2 homolog) rescues locomotion defects in HtrA2 mutants, mimicking Parkin-mediated mitophagy .
Experimental Design:
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Co-Immunoprecipitation (Co-IP): Use anti-HA/FLAG tags to pull down PINK1-HTRA2 complexes from mitochondrial lysates .
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Phosphomimetic Mutants: Generate S142D/S212D variants to test phosphorylation-dependent activation .
What are the critical controls for RNAi-mediated HTRA2 knockdown studies?
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Off-Target Validation: Use two independent RNAi lines (e.g., VDRC #12345, #67890) to confirm phenotype consistency .
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Rescue Experiments: Co-express RNAi-resistant HtrA2 cDNA under UAS control .
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Mitochondrial Markers: Include MitoTracker Red and ATP5A antibodies to distinguish apoptosis from mitochondrial dysfunction .
How to design mutagenesis studies to investigate HTRA2's dual regulatory switches?
HTRA2’s activity is regulated by N-terminal ligand binding and PDZ domain allostery . Key targets:
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Allosteric Mutants: Delete PDZ domain (ΔPDZ) or disrupt N-terminal AVPS motif (Δ1-15) to uncouple activation steps .
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Protease-Deficient Mutants: Replace catalytic Ser306 with Ala (S306A) to abolish enzymatic activity .
Functional Readouts:
| Mutant | Substrate Cleavage | XIAP Binding (SPR) | Mitochondrial Localization |
|---|---|---|---|
| WT | 100% | Yes (KD = 5nM) | Yes |
| ΔPDZ | 220% | No | Yes |
| S306A | 0% | Yes | Yes |
| Δ1-15 | 40% | No | No (cytosolic) |
