CTSZ Mouse, Active

What is the basic structure and properties of CTSZ Mouse, Active protein?

CTSZ Mouse Recombinant is a single, glycosylated, polypeptide chain containing 292 amino acids (residues 23-306) with a molecular mass of 32.8kDa. The recombinant protein is typically fused to an 8 amino acid His tag at the C-terminus and purified using proprietary chromatographic techniques . For experimental applications, CTSZ is often formulated in a solution containing Phosphate Buffered Saline (pH 7.4) with 10% glycerol to maintain stability .

CTSZ belongs to the peptidase C1 family and exhibits both carboxy-monopeptidase and carboxy-dipeptidase activities. These enzymatic capabilities distinguish it functionally from other cathepsins and contribute to its specialized biological roles .

What are the common synonyms and alternative designations for CTSZ?

When reviewing literature and sourcing reagents, researchers should be aware of multiple nomenclature variations:

These alternative designations appear throughout the literature and in research catalogs, which can sometimes lead to confusion when comparing studies .

What are the optimal storage conditions for maintaining CTSZ Mouse, Active protein viability?

For optimal preservation of CTSZ Mouse Active protein, storage conditions should be selected based on intended usage timeframe:

• Short-term usage (2-4 weeks): Store at 4°C if the entire vial will be used within this period .

• Long-term storage: Store frozen at -20°C. For extended preservation periods, it is strongly recommended to add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) .

Critical considerations for maintaining protein stability include:

• Avoiding multiple freeze-thaw cycles, which can significantly degrade protein activity

• Using appropriate carrier proteins to prevent adsorption to surfaces and maintain conformational stability

• Storing in small aliquots to minimize freeze-thaw events

What protein extraction protocols are recommended for CTSZ detection in mouse tissue samples?

Based on established research methodologies, the following extraction protocol is recommended for optimal CTSZ recovery from mouse tissue samples:

For protein extraction from lung and spleen homogenates:

• Perform initial PBS wash of tissue samples

• Extract protein using RIPA buffer supplemented with 1X Protease Inhibitor Cocktail (Sigma-Aldrich)

• For Trizol-stored samples:

  • Homogenize samples with sterile beads at 4.5 m/s for 30s using FastPrep-24 Homogenizer

  • Precipitate protein for 15 minutes using 9 volumes of 100% methanol at room temperature

  • Centrifuge protein precipitate at 3000rpm for 5 minutes

  • Dry precipitate for 5 minutes

  • Wash in equal volume of 90% methanol

  • Centrifuge for 1 minute at 3000rpm

  • Dry for 10 minutes

  • Resuspend in 1mL of RIPA buffer with 1X Protease Inhibitor Cocktail

  • Heat for 5-10 minutes at 95°C

For SDS-PAGE analysis:

• Combine equal volumes of sample with Laemmli Sample Buffer and 2-Mercaptoethanol

• Heat at 95°C for 5 minutes

• Separate proteins using 4-20% Mini-PROTEAN TGX Stain-Free Protein Gel

How can researchers effectively detect CTSZ protein in experimental samples?

For reliable detection of CTSZ in experimental samples, Western blotting has proven most effective with the following methodology:

• Transfer separated proteins to a polyvinylidene fluoride (PVDF) membrane using a semi-dry transfer protocol

• Block membrane using EveryBlot Blocking Buffer (BioRad)

• Perform primary antibody staining at 4°C overnight using Human/Mouse/Rat Cathepsin X/Z/P Antibody (R&D Systems; AF934) at a 1:2000 dilution in blocking buffer

This approach has been validated in studies examining CTSZ expression across different mouse strains, including comparison between wild-type and knockout models .

What role does CTSZ play in tuberculosis susceptibility and pathogenesis?

Recent research has established CTSZ as a conserved susceptibility factor in tuberculosis (TB) infection. The relationship between CTSZ and TB susceptibility has been demonstrated through multiple lines of evidence:

• Genetic variation: Collaborative Cross (CC) strains harboring the susceptible NOD Tip5 locus, which contains a hypomorphic variant of Ctsz, show reduced CTSZ protein levels compared to resistant C57BL/6 (B6) mice .

• Knockout validation: Ctsz−/− mice exhibit:

  • Significantly increased pulmonary Mycobacterium tuberculosis (Mtb) burden at multiple timepoints (2 weeks: 4.09 log₁₀CFU vs. 3.41 in B6; 4 weeks: 5.17 log₁₀CFU vs. 4.09 in B6; p<0.05)

  • Earlier bacterial dissemination to the spleen

  • Significantly reduced survival time following aerosol infection (p=0.008)

• Inflammatory signature: Ctsz−/− mice show a distinctive cytokine profile characterized by:

  • Elevated levels of TH1-associated cytokines (TNF-α, IL-1β)

  • Reduced levels of GM-CSF, IL-6, LIF, and VEGF compared to wild-type mice

  • Significantly higher CXCL1 production (a biomarker of active TB) throughout infection

These findings position CTSZ as a critical determinant of TB disease outcomes and suggest that genetic variation in this protein affects susceptibility to mycobacterial infection.

How does CTSZ influence tumor metastasis in cancer models?

CTSZ has been implicated in tumor metastasis through several interconnected mechanisms:

• Epithelial-Mesenchymal Transition (EMT): Overexpression of CTSZ in cancer cell models (QGY-7703 cells) induces EMT by:

  • Downregulating epithelial markers (E-cadherin, α-catenin, and β-catenin)

  • Upregulating mesenchymal markers (N-cadherin, fibronectin, and vimentin)

• Cytoskeletal reorganization: CTSZ overexpression promotes formation of filopodia, cellular protrusions that facilitate migration and invasion .

• Matrix Metalloproteinase (MMP) regulation: CTSZ significantly upregulates several MMPs involved in extracellular matrix degradation:

  • MMP2, MMP3, and MMP9 expression increases following CTSZ overexpression

  • Both total MMP9 and the active form (75kDa) increase in CTSZ-overexpressing cells

The combined effect of these changes creates a cellular phenotype with enhanced invasive and migratory capabilities, explaining the association between CTSZ expression and metastatic potential in various cancer types.

How can CTSZ knockout mouse models be validated for experimental studies?

Validation of CTSZ knockout models requires comprehensive assessment at multiple levels:

• Genotypic validation:

  • PCR-based genotyping to confirm targeted disruption of the Ctsz gene

  • Sequencing of the targeted region to verify the exact nature of the genetic modification

• Protein-level validation:

  • Western blot analysis using specific anti-CTSZ antibodies to confirm absence of CTSZ protein

  • Comparison with wild-type controls to ensure complete protein elimination

  • Verification in multiple tissues (e.g., lung, spleen) where CTSZ is normally expressed

• Functional validation:

  • Assessment of physiological parameters influenced by CTSZ (e.g., cytokine profiles)

  • Baseline characterization in uninfected state

  • Challenge with disease models known to be influenced by CTSZ function (e.g., Mtb infection)

Recent studies have successfully validated Ctsz−/− models by demonstrating absence of CTSZ protein in lung and spleen homogenates and confirming altered susceptibility to Mtb infection, providing a methodological framework for future research .

What are the key considerations when designing migration and invasion assays to assess CTSZ function?

When investigating CTSZ's role in cell migration and invasion, careful experimental design is essential:

• Cell invasion assay:

  • Use precoated cell invasion kits (e.g., Chemicon International)

  • Fix invaded cells with methanol and stain with crystal violet

  • Capture images from 3 randomly selected fields

  • Count cells that have invaded through the extracellular matrix

  • Perform at least three independent experimental repeats for statistical validity

• Wound healing assay:

  • Culture cells to 90% confluency

  • Create a standardized wound using sterile tips

  • Take pictures from identical areas at 0, 12, and 24 hours after scraping

  • Measure wound closure rates quantitatively

• Controls and variables to consider:

  • Include both wild-type and vector-only controls

  • Generate multiple independent clones expressing CTSZ (e.g., CTSZ-C1, CTSZ-C2)

  • Verify CTSZ expression levels in all experimental groups via Western blot

  • Consider the influence of cell proliferation rates on apparent migration

How do different genetic backgrounds influence CTSZ expression and function in mouse models?

Genetic background significantly impacts CTSZ expression and function, with important implications for experimental design:

• Strain-specific variation:

  • Collaborative Cross (CC) strains harboring the NOD Tip5 variant (CC033, CC038) exhibit significantly lower baseline levels of CTSZ protein compared to C57BL/6 mice

  • This reduced expression correlates with increased susceptibility to Mtb infection

• Sex-dependent effects:

  • In survival studies following Mtb infection, the mortality risk associated with CTSZ ablation was predominantly driven by male mice

  • This suggests hormonal or sex-linked genetic factors may influence CTSZ function

• Experimental implications:

  • Researchers should carefully consider background strain when designing experiments

  • Sex matching is critical for accurately interpreting results

  • Studies should include appropriate genetic controls (e.g., littermates, backcrossed strains)

These considerations highlight the importance of standardizing genetic backgrounds when studying CTSZ function across different experimental contexts.

How does CTSZ regulate inflammatory cytokine production in infection models?

CTSZ plays a critical role in regulating inflammatory cytokine production, particularly CXCL1, through mechanisms that appear to be both infection-dependent and infection-independent:

• Elevated baseline CXCL1 production:

  • Ctsz−/− mice show significantly higher lung CXCL1 levels even in the uninfected state compared to wild-type mice (p=0.007)

  • This suggests CTSZ constitutively suppresses CXCL1 production

• Enhanced CXCL1 response during infection:

  • Following Mtb infection, Ctsz−/− mice exhibit significantly elevated CXCL1 levels throughout the course of infection (2-4 weeks)

  • This enhanced production occurs independently of bacterial burden differences

• Macrophage-specific effects:

  • Bone marrow-derived macrophages (BMDMs) from Ctsz−/− mice rapidly induce CXCL1 following mycobacterial infection

  • Transcriptional data across species (mice, humans, macaques, zebrafish) indicates cathepsin Z expression is highest in macrophages following infection

The mechanism appears to involve CTSZ functioning as a negative regulator of CXCL1 production, potentially through proteolytic processing of signaling molecules or transcription factors involved in CXCL1 expression.

What molecular mechanisms underlie CTSZ-mediated epithelial-mesenchymal transition in cancer models?

CTSZ drives epithelial-mesenchymal transition (EMT) through coordinated regulation of multiple molecular pathways:

• Reprogramming of cell adhesion molecules:

  • Downregulation of epithelial markers:

    • E-cadherin (cell-cell adhesion)

    • α-catenin and β-catenin (adherens junction components)

  • Upregulation of mesenchymal markers:

    • N-cadherin (promotes motility)

    • Fibronectin (extracellular matrix component)

    • Vimentin (intermediate filament protein)

• Cytoskeletal reorganization:

  • CTSZ overexpression induces formation of filopodia, actin-rich plasma membrane protrusions important for cell migration

  • These structural changes were visualized by F-actin staining with fluorescein isothiocyanate conjugated phalloidin

• Extracellular matrix remodeling:

  • CTSZ significantly upregulates matrix metalloproteinases:

    • MMP2, MMP3, and MMP9 mRNA expression increases

    • Active form of MMP9 protein increases

  • These MMPs degrade extracellular matrix components, facilitating invasion

The combined effect of these molecular changes transforms epithelial cells toward a more mesenchymal, migratory, and invasive phenotype, explaining CTSZ's contribution to cancer progression.

How does mouse CTSZ research translate to understanding human disease?

Research on mouse CTSZ has significant translational relevance to human disease, particularly in tuberculosis and cancer:

• Tuberculosis:

  • Five variants in human CTSZ have been identified as correlates of TB severity in a Ugandan patient cohort

  • CTSZ protein has been detected in CD68+ macrophages within human pulmonary granulomas

  • This localization places CTSZ at the host-pathogen interface in human TB

• Cancer:

  • CTSZ is expressed ubiquitously in human cancer cell lines and primary tumors

  • Similar to mouse models, human CTSZ plays a role in tumorigenesis and metastasis

  • The molecular mechanisms identified in mouse models (EMT induction, MMP upregulation) appear conserved in human cancers

• Cross-species conservation:

  • Transcriptional data from multiple species (mice, humans, macaques, zebrafish) show conserved patterns of cathepsin Z expression

  • CTSZ is consistently highly expressed in macrophages following infection across species

  • This conservation suggests fundamental biological roles that transcend species boundaries

These correlations support the value of mouse CTSZ research as a model for understanding human disease mechanisms and identifying potential therapeutic targets.

What potential therapeutic applications exist based on CTSZ research findings?

Current research on CTSZ suggests several promising therapeutic applications:

• Tuberculosis interventions:

  • CTSZ augmentation strategies could potentially enhance TB resistance

  • Targeting the CTSZ-CXCL1 axis might reduce inflammatory damage during infection

  • Genetic screening for CTSZ variants could identify high-risk individuals for preventative intervention

• Cancer therapeutics:

  • CTSZ inhibitors could potentially reduce tumor metastasis by:

    • Preventing EMT induction

    • Blocking MMP upregulation

    • Reducing filopodia formation

  • Such approaches might be particularly effective in cancers with CTSZ overexpression

• Biomarker applications:

  • CTSZ expression levels could serve as prognostic indicators in certain cancers

  • The CTSZ-regulated cytokine CXCL1 might function as a biomarker for disease progression in tuberculosis

These potential applications highlight the clinical relevance of ongoing CTSZ research and suggest avenues for translating basic science findings into therapeutic interventions.