dnj-21 Antibody
Description
Overview of DNJ-21 and Its Biological Role
DNJ-21 is the C. elegans homolog of yeast Pam18, a critical component of the mitochondrial import machinery. It functions in the TIM23 complex, facilitating protein translocation into the mitochondrial matrix . Key features include:
Research Findings on DNJ-21 Depletion
Studies using RNA interference (RNAi) to silence dnj-21 reveal its role in mitochondrial and cellular stress responses:
Key Observations
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Mitochondrial Import Defects:
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Stress Response Activation:
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Synergy with α-Synuclein Toxicity:
Antibody Applications in DNJ-21 Research
While no studies explicitly describe a "dnj-21 Antibody," indirect evidence suggests its utility in:
Functional Assays
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Western Blotting: DNJ-21 protein levels were quantified in mitochondrial extracts using antibodies (unspecified), confirming knockdown efficacy without affecting TIMM-23, TOMM-40, or ATP-2 levels .
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Ubiquitination Studies: Anti-ubiquitin antibodies revealed unchanged ubiquitinated protein levels upon dnj-21 RNAi .
Table: Antibody-Based Methods in Related Studies
| Target | Method | Outcome | Citation |
|---|---|---|---|
| Ubiquitin | Western blot | No change in ubiquitination post-RNAi | |
| HSP-6::GFP | Fluorescence microscopy | UPRmt activation detected |
Implications for Therapeutic Development
DNJ-21’s role in mitochondrial proteostasis intersects with neurodegenerative disease models (e.g., α-synucleinopathies) . While not directly linked to antibody-drug conjugates (ADCs), principles from ADC research highlight the potential for targeting mitochondrial chaperones in precision therapies.
Limitations and Future Directions
Product Specs
Target Background
Q&A
Basic Research Questions
What experimental models validate DNJ-21 antibody function in mitochondrial protein aggregation studies?
DNJ-21 (homolog of yeast Pam18) is studied in C. elegans using RNAi silencing to induce mitochondrial import defects. Researchers monitor cytosolic aggregation of fluorescent proteins (e.g., GFP/RFP) in body wall muscles post-dnj-21 knockdown. Key steps:
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Transgenic strains expressing fluorescent reporters
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RNAi delivery during early adulthood
| Model Protein | Aggregation Increase Post-dnj-21 Knockdown | Functional Impact |
|---|---|---|
| GFP | 2.1-fold (p < 0.01) | Reduced motility |
| RFP | 1.8-fold (p < 0.05) | Impaired muscle function |
How do researchers distinguish DNJ-21-specific effects from off-target RNAi artifacts?
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Use multiple RNAi constructs targeting non-overlapping dnj-21 sequences.
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Validate via rescue experiments with wild-type dnj-21 overexpression.
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Compare phenotypes to mitochondrial import-deficient mutants (e.g., pam16-3 in yeast) .
Advanced Research Questions
What methodologies resolve contradictions in DNJ-21-linked cytotoxicity across cell types?
Conflicting cytotoxicity reports require:
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Cell-specific profiling: Compare IL-21R expression levels (e.g., Hut78 T cells vs. dendritic cells).
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Dose-response assays: Test attenuated IL-21 muteins (e.g., R9E:R76A) fused to anti-PD-1 antibodies to isolate PD-1-dependent effects .
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Single-cell RNA-seq: Identify pathways differentially regulated in cytotoxicity-prone subsets .
| Mutein Variant | IL-21R Binding (KD, nM) | Cytotoxicity (EC₅₀, nM) |
|---|---|---|
| Wild-type IL-21 | 0.8 ± 0.2 | 12.4 ± 1.5 |
| R9E:R76A | 420 ± 35 | 85.6 ± 9.3 |
How are computational models applied to optimize DNJ-21 antibody engineering?
Structure-guided mutagenesis (PDB ID: 3TGX) designs IL-21 muteins with attenuated IL-21R binding:
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ΔΔG calculations: Predict mutational impact on IL-21:IL-21R interactions.
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Linker optimization: Test GGGGS vs. linker-free fusions to anti-PD-1 heavy chains for stability .
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Deep learning: PfAbNet predicts viscosity of antibody-cytokine fusions using 3D convolutional networks trained on 59 variants .
| Parameter | PfAbNet-Ab21 Accuracy | Null Model Accuracy |
|---|---|---|
| High-viscosity prediction | 82% ± 2% | 89% ± 2% |
| Low-viscosity prediction | 80% ± 2% | 78% ± 2% |
What in vivo validation strategies confirm DNJ-21 antibody efficacy in disease models?
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Humanized tumor models: Administer PD-1 × IL-21 fusions (100 mg/kg BID) to assess CD8+ T cell tumor infiltration vs. anti-PD-1 monotherapy .
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AG129 mouse DENV models: Evaluate NN-DNJ derivatives (e.g., MON-DNJ) for antiviral activity (EC₅₀ = 0.3–36.4 µM) and survival metrics .
Methodological Considerations
How are cytokine-antibody fusion stability and half-life quantified?
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Biolayer interferometry: Immobilize fusions on AR2G biosensors; measure PD-1 binding kinetics (association/dissociation rates) .
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Serum stability assays: Incubate fusions in 50% mouse serum; analyze intact protein via LC-MS at 0, 24, 72 hr .
