GSDMC Antibody

Advanced Research Questions

• How does GSDMC cleavage contribute to pyroptotic cell death mechanisms?

  GSDMC-mediated pyroptosis follows a precise molecular mechanism:

  1. Cleavage activation: GSDMC is cleaved primarily by caspase-8 at Asp240 in human GSDMC (or Asp233 in mouse GSDMC4), releasing the N-terminal domain (1-365 amino acids) .

  2. Pore formation mechanism: The liberated N-terminal domain:

     • Binds to lipid membranes (demonstrated by liposome binding assays)

     • Forms regular-shaped pores of consistent size in the membrane

     • Causes severe membrane leakage (validated through Tb³⁺ release assays)

     • Translocates from cytosol to cell membrane (observable by confocal microscopy)

  3. Cellular effects: GSDMC N-terminal domain-induced pyroptosis is characterized by:

     • Cell swelling with large bubbles forming on the cell surface

     • Increased lactate dehydrogenase (LDH) release into the medium

     • Detection of GSDMC (1-365) protein in the cell culture supernatant

  Research has shown the N-terminal domain alone is sufficient to trigger pyroptosis when expressed in cells, while full-length GSDMC remains inactive until cleaved . This mechanism parallels other gasdermin family members but with distinct upstream regulators and tissue distribution.

• What are the differences between GSDMC isoforms and how do they affect antibody selection?

  Multiple GSDMC isoforms exist with important functional and recognition differences:

  Human GSDMC isoforms:
  The canonical human GSDMC protein (UniProt ID: Q9BYG8) consists of 508 amino acids with a molecular weight of 58 kDa . While specific human isoforms are less characterized in the provided search results, differential antibody reactivity suggests potential variants.

  Mouse GSDMC isoforms:
  Mouse has four distinct GSDMC isoforms (mGSDMC1-4) with critical functional differences:

  • Only mGSDMC4 contains the critical Asp233 residue (equivalent to human Asp240) required for caspase-8 cleavage and pyroptotic activity

  • When tested, only mGSDMC4 could be cleaved following α-ketoglutarate (α-KG) treatment, while mGSDMC1-3 were resistant to this cleavage

  • mGSDMC4 knockdown alone was sufficient to impair DM-αKG-induced pyroptosis in B16 cells

  Antibody selection considerations:

  1. Epitope location: Select antibodies whose epitopes don't span cleavage sites if detecting both full-length and cleaved forms is desired

  2. Cross-reactivity: Some antibodies (e.g., ab225635) detect multiple mouse GSDMC isoforms with varying affinity

  3. Target region specificity: For studying pyroptosis, antibodies targeting the N-terminal domain (aa 1-100) may be preferable for detecting active fragments

  4. Validation in knockout models: Antibodies like ab225635 have been validated using mGSDMC1-4 knockout mouse tissues, confirming specificity

  When studying GSDMC in mouse models, researchers should be aware that antibodies may have differential reactivity across the four mGSDMC isoforms.

• What signaling pathways regulate GSDMC expression and activity in disease contexts?

  GSDMC expression and activation are regulated by several key signaling pathways with disease-specific importance:

  Hypoxia-PD-L1-STAT3 pathway in cancer:

  • Hypoxic stress induces nuclear translocation of PD-L1

  • Nuclear PD-L1 partners with phosphorylated STAT3 (p-Y705-STAT3)

  • This complex binds to the STAT3-binding site in the GSDMC promoter

  • Transcriptional activation of GSDMC occurs under hypoxic conditions

  • GSDMC is then cleaved by caspase-8, switching TNFα-induced apoptosis to pyroptosis

  This pathway can be disrupted by:

  • PD-L1-NLS mutation (preventing nuclear localization)

  • STAT3-Y705F mutation (preventing phosphorylation)

  • Importin α/β inhibitors like ivermectin

  • STAT3 inhibitors like HO-3867

  α-ketoglutarate (α-KG) signaling:

  • The metabolite α-KG induces pyroptosis through caspase-8-mediated cleavage of GSDMC at Asp240 (human) or Asp233 (mouse GSDMC4)

  • This pathway represents a metabolic control of pyroptotic cell death

  • α-KG-induced GSDMC cleavage-dependent pyroptosis contributes to the repression of tumor growth and metastasis

  Type 2 immunity in intestinal epithelium:

  • IL-4/IL-13 treatment dramatically upregulates Gsdmc genes (Gsdmc2, Gsdmc3, Gsdmc4)

  • Helminth infection similarly induces Gsdmc expression

  • This upregulation correlates with increased lytic cell death

  • Suggests GSDMC involvement in anti-parasitic immune responses

  Understanding these regulatory pathways provides potential intervention points for manipulating GSDMC-mediated pyroptosis in disease contexts.

• How can researchers effectively validate GSDMC antibody specificity?

  Rigorous validation of GSDMC antibody specificity requires multiple complementary approaches:

  Genetic validation methods:

  1. Knockout/knockdown controls:

     • Use GSDMC knockout tissues/cells as negative controls (as shown with mGSDMC1-4 knockout mouse tissues)

     • Apply siRNA or shRNA knockdown of GSDMC with subsequent Western blot to confirm signal reduction

     • Multiple research groups have employed knockdown approaches to validate antibody specificity

  2. Overexpression validation:

     • Transfect cells with GSDMC expression vectors alongside empty vector controls

     • Compare band intensities and positions between transfected and non-transfected samples

     • HEK-293T cells are commonly used for this purpose due to low endogenous expression

  Biochemical validation methods:

  1. Peptide competition assays:

     • Pre-incubate antibody with the immunizing peptide before application

     • Signal should be abolished or significantly reduced if antibody is specific

  2. Multiple antibody approach:

     • Use antibodies raised against different epitopes of GSDMC

     • Compare banding patterns across different antibodies

     • Consistent detection suggests higher specificity

  Tissue/cellular validation:

  1. Known expression patterns:

     • Verify detection in tissues with established GSDMC expression (trachea, spleen)

     • Check for expected cellular localization (primarily cytoplasmic until cleaved)

  2. Functional correlations:

     • Verify increased detection following known activators (e.g., α-KG treatment, hypoxia)

     • Confirm detection of cleavage products during pyroptosis

  Proper validation should include multiple methods, with genetic approaches (particularly knockout controls) providing the strongest evidence for specificity.

• What is the relationship between GSDMC expression and disease progression in different pathological conditions?

  GSDMC exhibits context-dependent roles across different diseases:

  GSDMC in cancer progression:

  • Renal cell carcinoma (KIRC): GSDMC is highly expressed in both metastatic cell lines (Caki-1, ACHN) and non-metastatic cell lines (Caki-2, A498) compared to normal renal tubular cells (HK2). Immunohistochemical analyses confirm significantly higher expression in KIRC tissues than in paired normal tissues. This overexpression correlates with poor survival, suggesting an oncogenic role .

  • Colorectal cancer: GSDMC functions as an oncogene. Silencing GSDMC leads to significant reduction in proliferation and tumorigenesis of colorectal cancer cells .

  • Gastric and esophageal carcinogenesis: Contrary to its role in colorectal cancer, GSDMC has been reported to play a tumor-suppressive role in these cancers .

  • Breast cancer: Under hypoxic conditions, PD-L1-induced GSDMC can be cleaved by caspase-8 to switch TNFα-induced apoptosis to pyroptosis .

  GSDMC in inflammatory diseases:

  • Psoriasis: GSDMC concentration is significantly higher in serum of psoriasis patients compared to controls. Psoriatic lesions exhibit significantly higher expression of GSDMC than non-lesional skin or control skin. This suggests potential involvement in the inflammatory processes of psoriasis .

  Disease Context	GSDMC Expression	Correlation	Potential Role
  Renal Cell Carcinoma	Increased	Poor survival	Oncogenic
  Colorectal Cancer	Increased	Enhanced tumorigenesis	Oncogenic
  Gastric/Esophageal Cancer	Variable	Reduced malignancy	Tumor suppressive
  Psoriasis	Increased in lesions and serum	Disease severity	Biomarker of hypoxia or cell proliferation

  These divergent roles highlight the importance of context-specific analysis when studying GSDMC in disease progression.

• How does GSDMC function compare to other gasdermin family members in pyroptotic pathways?

  The gasdermin family includes several members with both shared and distinct features in pyroptotic pathways:

  Structural and functional similarities:

  • All gasdermins share a two-domain architecture with an N-terminal pore-forming domain and a C-terminal inhibitory domain

  • Cleavage between domains releases the N-terminal portion that forms membrane pores

  • Pore formation is the common executioner mechanism leading to pyroptotic cell death

  GSDMC-specific characteristics:

  • Cleavage site: Human GSDMC is primarily cleaved at Asp240 by caspase-8, while mouse GSDMC4 is cleaved at the equivalent Asp233

  • Upstream activators: GSDMC activation is uniquely regulated by:

    • α-ketoglutarate (α-KG) metabolic signaling

    • Hypoxia-induced PD-L1/STAT3 pathway

    • IL-4/IL-13 signaling in intestinal type 2 immune responses

  • Tissue distribution: Predominantly expressed in trachea and spleen, with specific expression in epithelial tissues

  Comparison with other family members:

  Gasdermin	Primary Activator	Cleavage Site	Major Tissue Expression	Key Role
  GSDMC	Caspase-8	Asp240 (human)	Trachea, spleen, GI tract	Type 2 immunity, cancer context-dependent
  GSDMD	Caspase-1/4/5/11	Asp275 (human)	Immune cells	Canonical inflammasome pyroptosis
  GSDME	Caspase-3	Asp270 (human)	Cochlea, various epithelia	Apoptosis to pyroptosis switch
  GSDMB	Granzyme A	Lys229/Lys244	Esophagus, GI, immune cells	Cytotoxic lymphocyte-mediated pyroptosis

  Unlike GSDMD, which is central to inflammasome-mediated pyroptosis, GSDMC appears to function in more specialized contexts, including type 2 immunity and hypoxic tumor microenvironments .

• What methodological approaches are recommended for studying GSDMC-mediated pyroptosis?

  Several methodological approaches can be employed to study GSDMC-mediated pyroptosis:

  1. Pyroptotic cell death assessment:

  • LDH release assay: Measures lactate dehydrogenase released from pyroptotic cells into culture media

  • Propidium iodide (PI) uptake: Quantifies membrane permeabilization using flow cytometry

  • Morphological analysis: Observation of cell swelling with large bubbles forming on the cell surface, characteristic of pyroptosis

  2. GSDMC cleavage detection:

  • Western blotting: Use antibodies detecting both full-length (~58 kDa) and N-terminal fragments (~35 kDa)

  • In vitro cleavage assay: Incubate recombinant GSDMC with purified caspase-8 to demonstrate direct cleavage

  • Site-directed mutagenesis: Create D240A mutants (human) or D233A mutants (mouse GSDMC4) to confirm cleavage site specificity

  3. Pore formation analysis:

  • Liposome leakage assays: Mix cleaved GSDMC with liposomes and measure Tb³⁺ release

  • Electron microscopy of liposomes: Visualize pore formation on liposome surfaces

  • Membrane localization: Use confocal microscopy to track GSDMC N-terminal domain translocation to cell membranes

  4. Functional studies:

  • GSDMC knockdown/knockout: Use siRNA, shRNA, or CRISPR-Cas9 to deplete GSDMC and assess impact on pyroptosis

  • Domain expression: Express GSDMC N-terminal domain (aa 1-365) alone to demonstrate its pyroptotic activity

  • Inhibitor studies: Test caspase inhibitors (e.g., Z-VAD) to confirm the role of caspases in GSDMC activation

  5. Physiological context evaluation:

  • α-KG treatment: Induce GSDMC-dependent pyroptosis with α-KG or dimethyl-α-KG (DM-αKG)

  • Hypoxia models: Study GSDMC activation under hypoxic conditions to evaluate the PD-L1/STAT3 pathway

  • Type 2 immunity model: Use IL-4/IL-13 treatment or helminth infection models to study GSDMC in mucosal immunity

  These complementary approaches provide a comprehensive toolkit for investigating GSDMC-mediated pyroptosis in different experimental contexts.

• How does the subcellular localization of GSDMC change during pyroptotic activation?

  GSDMC undergoes dynamic subcellular relocalization during pyroptotic activation:

  1. Inactive state localization:

  • Full-length GSDMC resides primarily in the cytosol

  • Confocal microscopy shows diffuse cytoplasmic distribution of full-length GSDMC

  • The C-terminal domain acts as an inhibitor, preventing membrane binding

  2. Activation-induced relocalization:

  • Upon caspase-8-mediated cleavage at Asp240 (human) or Asp233 (mouse GSDMC4):

    • The N-terminal fragment (aa 1-365) is released from inhibition

    • This fragment rapidly translocates to the plasma membrane

    • Membrane association is driven by affinity for specific lipids

  3. Membrane pore formation:

  • At the membrane, GSDMC N-terminal domains:

    • Oligomerize to form multimeric structures

    • Create pores of consistent diameter (visible by electron microscopy in liposome models)

    • Alter membrane permeability, allowing ion and small molecule exchange

  4. Terminal events:

  • In late-stage pyroptosis:

    • GSDMC N-terminal fragments can be detected in cell culture supernatants

    • This coincides with complete membrane rupture and cell lysis

  Visualization methods:

  • Use N-terminal specific antibodies for tracking active fragments

  • Employ fluorescently-tagged GSDMC constructs (N-terminal tags preferred)

  • Apply immunofluorescence with antibodies targeting different epitopes to distinguish full-length from cleaved forms

  • Implement fractionation techniques to separate cytosolic from membrane fractions for biochemical analysis

  This dynamic relocalization is a defining feature of GSDMC activation and is critical for its pyroptotic function.

• What are the emerging therapeutic implications of targeting GSDMC in disease?

  Research on GSDMC has revealed several potential therapeutic applications:

  Cancer treatment approaches:

  • Tumor suppression: α-KG-induced GSDMC cleavage-dependent pyroptosis contributes to the repression of tumor growth and metastasis, suggesting α-KG or mimetics as potential therapeutic agents .

  • Context-dependent targeting: Given GSDMC's divergent roles in different cancers (oncogenic in colorectal cancer, tumor-suppressive in gastric cancer), cancer-specific approaches are necessary .

  • Combination with immunotherapy: The PD-L1/STAT3/GSDMC axis suggests potential synergy with immune checkpoint inhibitors in certain cancer contexts .

  Inflammatory disease management:

  • Psoriasis biomarker: Elevated serum GSDMC and decreased urinary GSDMC/creatinine ratio could serve as non-invasive biomarkers for psoriasis monitoring .

  • Anti-inflammatory approaches: Targeting GSDMC activation might reduce pathological inflammation in conditions with excessive pyroptosis.

  Parasitic infection control:

  • Type 2 immunity enhancement: GSDMC's role in intestinal immune responses to helminths suggests potential applications in enhancing anti-parasitic immunity .

  • Anti-helminthic strategies: The release of anti-parasitic factors through GSDMC-mediated pyroptosis could be leveraged for treating helminth infections .

  Therapeutic targeting strategies:

  1. Direct inhibition: Design of cleavage-site specific inhibitors to prevent GSDMC activation

  2. Upstream modulation: Targeting regulatory pathways (PD-L1/STAT3, α-KG metabolism)

  3. Selective activation: Controlled induction of GSDMC-mediated pyroptosis in cancer cells

  4. Biomarker utilization: Use of GSDMC expression/cleavage as a companion diagnostic

  While these therapeutic approaches show promise, their development requires further understanding of GSDMC regulation and function in specific disease contexts.

• What are the critical considerations for optimizing immunohistochemical detection of GSDMC in tissue samples?

  Optimizing immunohistochemical detection of GSDMC requires attention to several critical factors:

  1. Fixation and tissue processing:

  • Fixative selection: 10% neutral buffered formalin is typically recommended

  • Fixation duration: Optimal fixation time is 12-24 hours; overfixation can mask epitopes

  • Section thickness: 4-5 μm sections provide optimal staining without excessive background

  2. Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0 has been shown to be most effective

  • Alternative approach: Citrate buffer pH 6.0 can be used if TE buffer yields suboptimal results

  • Retrieval duration: 15-20 minutes at high pressure or 95-98°C is typically sufficient

  3. Antibody selection and optimization:

  • Dilution range: Most GSDMC antibodies perform optimally between 1:50-1:500 for IHC

  • Incubation parameters: Overnight incubation at 4°C often yields superior results compared to shorter incubations

  • Detection systems: Polymer-based detection systems may provide better signal-to-noise ratio than avidin-biotin methods

  4. Validation controls:

  • Positive tissue controls: Human kidney tissue has been validated for GSDMC detection

  • Cell line controls: HeLa and HepG2 cells are suitable for validating antibody reactivity

  • Negative controls: Include primary antibody omission and, when available, GSDMC knockout tissues

  5. Interpretation guidelines:

  • Localization pattern: Primarily cytoplasmic staining is expected for full-length GSDMC

  • Distribution patterns: Differentiated epithelial cells typically show stronger expression

  • Pathological contexts: Expression may be significantly upregulated in certain conditions (e.g., psoriatic lesions)

  6. Quantification approaches:

  • Scoring systems: Use established semi-quantitative systems (H-score or Allred score)

  • Digital pathology: Consider image analysis software for more objective quantification

  • Correlation analysis: Compare IHC results with other detection methods (e.g., Western blot) when feasible

  Following these guidelines will help ensure reliable and reproducible GSDMC detection in tissue samples for research applications.