Cleaved-CASP6 (D179) Antibody
Product Specs
Target Background
- The prodomain region of caspase-6 was found to be intrinsically disordered regardless of its activation state. However, complete removal of this region resulted in the protection of the adjacent 26-32 region, suggesting that this region might play a regulatory role. Understanding the molecular dynamics of caspase-6 in solution provides a comprehensive framework for designing therapeutic approaches to neurodegenerative disorders. PMID: 28864531
- SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Huntington's and Alzheimer's diseases. PMID: 28659495
- Research indicates that caspase-6 activity in the anterior olfactory nucleus of the olfactory bulb reflects degeneration in the entorhinal cortex. This suggests that caspase-6 activity in the olfactory bulb could be associated with cognitive decline and early Alzheimer's disease. PMID: 27931265
- Findings support the possibility that caspase-6 deactivating mutations may contribute to multifactorial carcinogenic transformations. PMID: 28726391
- Caspase-6 undergoes a helix-strand transition upon substrate binding. It exhibits distinct conformational dynamics in its 130's region, with local pKa values of key amino acid residues within this region varying between the unliganded (helical) and VEID-bound (strand) states of caspase-6. PMID: 28154009
- Following specific binding and internalization into HER2-overexpressing tumor cells, the e23sFv-Fdt-casp6 protein induced tumor cell apoptosis and inhibited the proliferation of HER2-overexpressing A172 and U251MG cells in vitro, but not in U87MG cells with undetectable HER2 levels. PMID: 27633091
- Studies have identified novel members of the CASP6 interactome, demonstrating that several of them are involved in key signaling pathways observed in neurodegenerative diseases. PMID: 26908611
- The ability of sox11 to reduce effector caspase activity was reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also possessed the ability to reduce caspase-6 activity, albeit to a lesser extent than sox11. PMID: 26505998
- Caspase-6 plays a role in activating caspase-3 in Tau truncation. PMID: 24363090
- p53 activity is a crucial upstream regulator of caspase-6 activity in Huntington's disease. PMID: 24070868
- In a study, the crystal structure of a full-length CASP6 zymogen mutant, proCASP6H121A, was solved. PMID: 24419379
- Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. PMID: 24265764
- Significant associations have been found between CpG sites and patient sex, including DNA methylation in CASP6, a gene that may respond to estradiol treatment, and in HSD17B12, which encodes a sex steroid hormone. PMID: 24058506
- Caspase 6 activity in the entorhinal cortex identifies aged individuals at risk for developing Alzheimer's disease. PMID: 23402898
- Research demonstrates that in the absence of caspase 6 activity, intrinsic triggers of apoptosis induce the receptor-interacting-kinase-1-dependent production of pro-inflammatory cytokines. PMID: 22858542
- Zinc binding at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation. PMID: 22891250
- A peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site. This peptide acts via a novel allosteric mechanism that promotes caspase tetramerization. PMID: 22683611
- Results revealed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a novel strategy of rational CASP6 drug design. PMID: 22433863
- Caspase-6 cleaves human TERT at residues E129 and D637 as part of the apoptosis pathway in cultured cells. PMID: 21936563
- A new crystal form of apo-caspase-6 is presented in its canonical conformation, identifying the previous apostructure as a hydrogen-ion concentration (pH)-inactivated form of caspase-6. PMID: 21621544
- Data suggests that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that more closely resembles canonical caspases. PMID: 21111746
- CASP6 can be activated and regulated through intramolecular self-cleavage. PMID: 20890311
- Intact IRAK-M is strongly expressed in resting alveolar macrophages but is cleaved in patients with pneumonia via neutrophil-mediated induction of CASP-6 activity. PMID: 21098228
- These results suggest that pro-Casp6b could negatively regulate pro-Casp6a activation in neurons and prevent Casp6a-mediated axonal degeneration. PMID: 20682790
- The NPM mutant specifically inhibits the activities of the cell-death proteases, caspase-6 and -8, through direct interaction with their cleaved, active forms, but not the immature procaspases. PMID: 20606168
- Results indicate that p97 is cleaved by Casp6 in Alzheimer's disease, suggesting p97 cleavage as an important mechanism for ubiquitin proteasome system impairment. PMID: 20427671
- p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. PMID: 19897582
- Research identified executioner caspase-6 as a transcriptional target of p53. The mechanism involves DNA binding by p53 to the third intron of the caspase-6 gene and transactivation. PMID: 12089322
- Pro-CASP6 was the only proenzyme whose localization was limited to the cytosol in U937 cells during TPA-induced differentiation. PMID: 12145703
- ARK5 negatively regulates procaspase-6 by phosphorylation at Ser257, leading to resistance to the FasL/Fas system. PMID: 15273717
- Programmed cell death was executed by caspase 6 in Streptococcus pneumoniae infected lung epithelium. PMID: 15321985
- CASP-6 cleaves the N terminus of tau in vitro at D13, a semicanonical and hitherto undescribed caspase cleavage site in tau. This suggests a role for caspase-6 and N-terminal truncation of tau during neurofibrillary tangle formation and Alzheimer's disease progression. PMID: 15356202
- Caspase 6 cleaves periplakin at an unconventional recognition site, amino acid sequence TVAD. PMID: 15654952
- Data indicates that the splitting of BL41-E95-A cells induces de novo synthesis of a protein involved in the activation of casp-6 and casp-8, which cleaves 5-LO. PMID: 16135563
- Data suggests that the CASP6 gene is occasionally mutated in gastric and colorectal carcinomas. This also suggests the possibility that deficiency of caspase-6 expression might contribute to the pathogenesis of gastric cancers. PMID: 16948818
- Caspase-6 was overexpressed in 52.9% of the 210 cases studied, showing predominantly cytoplasmic with some nuclear staining. PMID: 16977583
- Active caspase-6 could be an early instigator of neuronal dysfunction. PMID: 17392160
- Resveratrol exhibits converse dose-related effects on fluorouracil-evoked colon cancer cell apoptosis; the role of CASP6 is reported. PMID: 18497562
- Expression of caspase 6 and caspase 14 genes differed between normal skin of keloid-prone individuals and normal skin of keloid-resistant patients. PMID: 18762957
- Caspase 6 cleaves cyclin B1 during mitotic catastrophe. Mitotic and apoptotic functions may be linked by caspase-dependent processing of mitotic activators. PMID: 18820706
- During hypoxia in tube-forming endothelial cells, caspase-7 is responsible for chromatin condensation and nuclear fragmentation, while caspase-6 is responsible for DNA ladder formation. PMID: 19022247
- The crystal structure of caspase-6, a selective effector of axonal degeneration, has been determined. PMID: 19694615
- Casp-6 is activated in familial forms of Alzheimer's disease, as previously observed in sporadic forms. PMID: 19915487
Q&A
What does the Cleaved-CASP6 (D179) Antibody specifically detect?
The Cleaved-CASP6 (D179) antibody specifically detects endogenous levels of the fragment of activated Caspase-6 p18 protein that results from cleavage adjacent to the aspartic acid residue at position 179 (D179) . This antibody recognizes the neoepitope created when caspase-6 undergoes proteolytic processing during activation, making it a valuable tool for specifically detecting the activated form of caspase-6 rather than the zymogen form.
What is the molecular basis for caspase-6 activation and the importance of the D179 cleavage site?
Caspase-6 activation involves a complex process of proteolytic cleavage at multiple sites. Complete activation of caspase-6 typically requires processing at three key sites:
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TETD 23 - Removing the prodomain
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DVVD 179 - Cleaving between the large and small subunits
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TEVD 193 - Additional processing site
The D179 cleavage site is particularly significant as it separates the large subunit from the small subunit, which is a critical step in the formation of the active enzyme . After cleavage, the large and small subunits dimerize to form the active caspase-6 enzyme. Interestingly, research has shown that caspase-6 can undergo a conformational change and bind substrates even in the absence of complete cleavage, suggesting a unique activation mechanism compared to other executioner caspases .
How does caspase-6 activation differ from other executioner caspases?
Caspase-6 exhibits several distinctive features in its activation compared to other executioner caspases such as caspase-3 and caspase-7:
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Independent activation pathway: Caspase-6 can be activated independently of caspases-3 and -7, suggesting it may self-activate or be cleaved by other proteases .
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Conformational flexibility: Caspase-6 undergoes substantial conformational changes upon binding active-site ligands, with up to 18% loss of CD signal at 222 nm, compared to only 2.3% for caspase-7 .
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Partially cleaved complexes: Active caspase-6 can exist as heterodimers containing one fully cleaved monomer and one uncleaved monomer, forming "half-cleaved" complexes similar to what has been observed with caspase-7 .
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Stability characteristics: Cleaved caspase-6 with both the prodomain and linker present shows enhanced stability, suggesting these regions work together to influence the enzyme's structural properties .
What are the recommended applications and optimal dilutions for the Cleaved-CASP6 (D179) Antibody?
The Cleaved-CASP6 (D179) Antibody has been validated for multiple applications with specific recommended dilutions:
These dilutions should be optimized for specific experimental conditions and sample types .
How should researchers prepare samples for optimal detection of cleaved caspase-6?
For optimal detection of cleaved caspase-6 using the D179 antibody, consider the following sample preparation methods:
Cell Lysate Preparation:
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Induce apoptosis in cells using appropriate stimuli (e.g., anti-DR5 antibody, staurosporine)
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Perform hypotonic lysis while preserving protein integrity
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Include protease inhibitors to prevent additional non-specific proteolysis
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Ensure samples are fresh or properly stored at -80°C to maintain epitope integrity
Tissue Sample Preparation:
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Properly fix tissues using paraformaldehyde
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For paraffin-embedded sections, perform antigen retrieval to expose the D179 neoepitope
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Block endogenous peroxidases and non-specific binding sites
The antibody can detect cleaved caspase-6 in multiple sample types including cell culture systems, tissue extracts, and in vivo tumor models .
What cross-reactivity considerations should researchers be aware of?
The Cleaved-CASP6 (D179) antibody has been documented to react with human and rat species . When using this antibody, consider the following specificity considerations:
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Forms detected: The antibody detects multiple forms of cleaved caspase-6, including the mature p18 fragment and larger partially processed forms .
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Potential cross-reactivity: In complex samples, the antibody might detect other proteins with similar molecular weights. Validation through immunoprecipitation can help confirm specificity .
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Distinguishing from other caspases: While the antibody is specific for the D179 cleavage site of caspase-6, researchers should be aware that other caspases (particularly caspase-3 and caspase-7) share some sequence similarity and may be present in the same samples .
For critical applications, researchers should include specific controls such as caspase-6 knockout samples or blocking with the immunogenic peptide to validate antibody specificity .
How can this antibody be used to investigate the unique activation mechanisms of caspase-6?
The Cleaved-CASP6 (D179) antibody serves as a powerful tool for investigating caspase-6 activation mechanisms through several advanced approaches:
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Detecting partially cleaved complexes: Research has shown that caspase-6 exists in multiple partially cleaved complexes that productively bind substrates. The D179 antibody can detect these intermediates (30-36 kDa proteins) in addition to the fully cleaved p18 fragment .
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Tracking activation kinetics: By combining time-course experiments with D179 antibody detection, researchers can map the sequential appearance of different cleaved forms of caspase-6.
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Investigating activation independence: Studies have demonstrated that caspase-6 activation does not require active caspases-3/-7. Researchers can use the D179 antibody in conjunction with caspase-3/-7 inhibitors (like AB13) to explore this unique activation pathway .
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Exploring conformational changes: When combined with activity-based probes like LE22, the D179 antibody can help correlate the presence of specific cleaved forms with catalytic activity .
What insights can be gained about caspase-6 structure-function relationships?
The D179 antibody can reveal important insights about caspase-6 structure-function relationships:
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Dimeric structures: Research has shown that caspase-6 dimers can contain mixtures of cleaved and uncleaved monomers. Immunoprecipitation with the D179 antibody can pull down these heterodimeric complexes, allowing researchers to study their composition and activity .
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Domain interactions: Cleaved caspase-6 with both the prodomain and linker present is the most stable form, indicating that these regions act in concert to stabilize the protein. The D179 antibody can help track different structural variants in experimental systems .
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Substrate-induced conformational changes: Upon binding active-site ligands, caspase-6 undergoes significant conformational changes with loss of helical content. The D179 antibody can be used to immunoprecipitate these different conformational states for further analysis .
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Phosphorylation effects: Phosphorylation regulates assembly of the caspase-6 substrate-binding groove. The D179 antibody can help researchers track how phosphorylation at sites like S257 affects the processing at the D179 site .
How does this antibody facilitate research on caspase-6 in neurodegenerative diseases?
The Cleaved-CASP6 (D179) antibody has significant applications in neurodegenerative disease research:
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Alzheimer's disease studies: Active caspase-6 and tau cleaved by caspase-6 are present in neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer's disease brains. The D179 antibody enables researchers to track caspase-6 activation in brain tissue samples .
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Neuronal death mechanisms: By combining the D179 antibody with neuronal markers, researchers can investigate the relationship between caspase-6 activation and neuronal death in various disease models.
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Therapeutic target validation: The antibody can be used to assess the efficacy of potential caspase-6 inhibitors in preventing D179 cleavage, thereby validating caspase-6 as a therapeutic target .
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Biomarker development: Detection of cleaved caspase-6 (D179) in cerebrospinal fluid or blood could potentially serve as a biomarker for neurodegenerative processes.
Why might researchers observe unexpected bands in Western blot analysis?
When using the Cleaved-CASP6 (D179) antibody in Western blot applications, researchers might encounter several patterns of unexpected bands:
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Multiple high molecular weight bands (30-36 kDa): These likely represent partially processed forms of caspase-6 that have been cleaved at D23 but not completely processed at D179 or D193. These are legitimate forms of cleaved caspase-6 and not necessarily non-specific binding .
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Bands corresponding to full-length caspase-6: The antibody might detect full-length caspase-6 in a complex with cleaved forms, particularly in dimeric structures where one monomer is cleaved and the other is intact .
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Slower than expected migration: Caspase-6 variants lacking the intersubunit linker may migrate more slowly than expected on SDS-PAGE, possibly due to interactions with the gel matrix or differences in protein shape .
To distinguish specific from non-specific signals, researchers should:
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Include both positive controls (apoptotic cells) and negative controls
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Consider using caspase-6 knockout/knockdown samples
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Perform peptide competition assays with the immunogenic peptide
What are the optimal experimental conditions for detecting cleaved caspase-6 in different cellular compartments?
Cleaved caspase-6 can localize to different cellular compartments depending on the cell type and activation conditions. For optimal detection:
How can researchers distinguish between different cleaved forms of caspase-6?
To distinguish between different cleaved forms of caspase-6:
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Use of multiple antibodies: Combine the D179 antibody with antibodies targeting other cleavage sites (D23, D193) to track different processing intermediates.
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SDS-PAGE optimization: Use gradient gels (10-20%) to better separate the various cleaved forms of caspase-6, which can range from approximately 18 kDa (p18 subunit) to 36 kDa (partially processed forms) .
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Two-dimensional electrophoresis: This technique can separate proteins based on both molecular weight and isoelectric point, potentially distinguishing between different cleaved forms with similar molecular weights.
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Mass spectrometry validation: To definitively identify the exact cleavage products detected by the D179 antibody, researchers can perform immunoprecipitation followed by mass spectrometry analysis .
How is the Cleaved-CASP6 (D179) antibody advancing research on caspase-6 activation mechanisms?
Recent research utilizing the Cleaved-CASP6 (D179) antibody has yielded several important insights:
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Unique auto-activation pathway: Studies have revealed that caspase-6 can undergo auto-activation independently of caspases-3 and -7, challenging the traditional caspase cascade model. The D179 antibody has been instrumental in tracking this process .
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Conformational plasticity: Research has demonstrated that caspase-6 exhibits significant conformational changes upon substrate binding, with up to 18% loss in helical content, compared to only 2.3% for caspase-7. This suggests a unique structural flexibility that may be related to its diverse biological functions .
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Half-cleaved complexes: The discovery that active caspase-6 can exist as dimers containing one cleaved and one uncleaved monomer provides new understanding of how executioner caspases are regulated. The D179 antibody helps detect these complexes through immunoprecipitation techniques .
What emerging roles of caspase-6 are being investigated using this antibody?
Beyond the traditional role in apoptosis, researchers are using the Cleaved-CASP6 (D179) antibody to investigate several emerging functions of caspase-6:
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Neurodegeneration pathways: Active caspase-6 and tau cleaved by caspase-6 are found in neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer's disease brains, suggesting a role in neurodegeneration that extends beyond apoptosis .
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Allosteric regulation: Studies identifying allosteric inhibitors against active caspase-6 indicate complex regulatory mechanisms that may be targeted therapeutically. The D179 antibody helps validate these inhibitors by confirming their effects on caspase-6 processing .
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Phosphorylation-dependent regulation: Recent research has revealed that phosphorylation regulates assembly of the caspase-6 substrate-binding groove, with important implications for its activation and activity. The D179 antibody helps track how phosphorylation affects processing at this critical site .
What technical advances are enhancing detection of cleaved caspase-6?
Recent technical advances are improving the detection and characterization of cleaved caspase-6:
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Activity-based probes: Optimized activity-based probes like LE22 show enhanced potency and sensitivity compared to earlier probes like AB50. When combined with the D179 antibody, these tools provide complementary information about both the presence and activity of cleaved caspase-6 .
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Multi-parameter analysis: Combining the D179 antibody with other markers of apoptosis or neurodegeneration in multi-parameter flow cytometry or imaging allows for more comprehensive analysis of caspase-6 in complex biological processes.
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Three-dimensional structural studies: Using the D179 antibody to identify and isolate specific cleaved forms of caspase-6 is enabling more detailed structural studies, revealing that unliganded mature caspase-6 adopts a conformation distinct from that observed in other caspases .
