Recombinant Human Chloride intracellular channel protein 3 (CLIC3)

What is the structure and functional characteristics of CLIC3?

Recombinant Human CLIC3 is a 236 amino acid protein that belongs to the chloride intracellular channel family. The full-length protein has a molecular sequence beginning with Met1 and ending at Arg236. In its native form, CLIC3 demonstrates dual functionality:

• As a soluble protein, it catalyzes glutaredoxin-like thiol disulfide exchange reactions with reduced glutathione as an electron donor

• When inserted into membranes, it forms outwardly rectifying chloride ion channels

CLIC3 exists in different conformational states depending on the cellular environment, enabling it to transition between its soluble oxidoreductase activity and membrane-associated channel functions. For research applications, recombinant CLIC3 is typically produced in Escherichia coli expression systems with >95% purity and endotoxin levels <1 EU/μg, making it suitable for various experimental procedures including SDS-PAGE and HPLC .

How can researchers effectively express and purify recombinant CLIC3 for experimental use?

The most effective method for generating recombinant CLIC3 involves bacterial expression systems, particularly E. coli. The recommended procedure includes:

• Cloning and Expression Vector Construction:

  • Clone the human CLIC3 cDNA (encoding Met1-Arg236) into a suitable expression vector

  • Include a C-terminal 6-His tag for purification purposes

• Expression Conditions:

  • Transform the construct into an E. coli expression strain

  • Induce protein expression with IPTG at optimal temperature (typically 25-30°C)

  • Harvest cells after 4-6 hours of induction

• Purification Protocol:

  • Lyse cells under native conditions

  • Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Apply additional purification steps if needed (ion exchange, size exclusion)

  • Verify purity by SDS-PAGE and HPLC

For optimal results, researchers should ensure the final recombinant CLIC3 preparation contains minimal endotoxin (<1 EU/μg) and maintains >95% purity for downstream applications .

What are the optimal storage conditions for maintaining CLIC3 stability and activity?

To maintain the structural integrity and functional activity of recombinant CLIC3:

• Store lyophilized protein at -20°C for up to 12 months

• After reconstitution, store at -80°C in small aliquots to avoid repeated freeze-thaw cycles

• For working solutions, maintain at 4°C for no more than 1 week

• Include stabilizing agents such as 10% glycerol or 1mM DTT to preserve redox-sensitive properties

• Monitor protein activity periodically using functional assays to ensure viability

The redox-sensitive nature of CLIC3 makes it particularly susceptible to oxidative inactivation, so storage buffers should be optimized accordingly to preserve its glutaredoxin-like activity .

How can researchers effectively measure CLIC3 channel activity in experimental systems?

Measuring CLIC3 channel activity requires specialized electrophysiological approaches. Based on established protocols, the following methodology is recommended:

• Whole-Cell Patch-Clamp Recording:

  • Use cells expressing CLIC3 (either endogenous or recombinant)

  • Employ an EPC-10 patch-clamp amplifier or equivalent

  • Filter data at 2.9 kHz and digitize at 10 kHz

  • Use patch electrodes with 2-4 MΩ resistance when filled with pipette solution

• Solution Composition:

  • Pipette solution: 140 mM N-methyl-d-glucamine (NMDG), 14 mM HCl, 126 mM L-aspartic acid, 2 mM Na₂ATP, 5 mM MgCl₂, 10 mM HEPES, 1 mM EGTA (pH 7.3 with Tris)

  • Standard bathing solution: 145 mM NMDG, 145 mM HCl, 7 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES (pH 7.4 with Tris)

  • Low Cl⁻ bathing solution: 145 mM NMDG, 145 mM L-aspartic acid, 7 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES (pH 7.4 with Tris)

• Protocol and Analysis:

  • Apply voltage step pulses (500 ms from -100 to +100 mV in 20-mV increments) or ramp pulses (100 ms from -100 to +100 mV)

  • Measure steady-state currents averaged at 450-500 ms on the step pulses

  • Normalize currents to corresponding membrane capacitance

  • Use NPPB (chloride channel blocker) to verify specificity of currents

  • Compare current-voltage relationships in standard versus low Cl⁻ conditions

The unique outwardly rectifying profile of CLIC3 chloride currents (with conductance ranging from approximately 3-400 pS, similar to other CLIC family members) can be identified through these methods .

What techniques are most effective for studying CLIC3's role in redox regulation and glutathione-dependent processes?

To investigate CLIC3's oxidoreductase activity and glutathione-dependent functions:

• Quantitative Mass Spectrometry:

  • Incubate recombinant CLIC3 with target proteins (e.g., TGM2)

  • Compare wild-type CLIC3 with enzymatically inactive mutants (e.g., C22A)

  • Analyze the reduced status of specific cysteines in target proteins

  • Quantify changes in reduction levels of specific cysteine residues

• Glutaredoxin Activity Assays:

  • Use coupled spectrophotometric assays with glutathione and NADPH

  • Monitor the reduction of disulfide substrates

  • Compare activities of wild-type CLIC3 versus mutated variants

  • Assess the effects of pH and redox environment on activity

• Site-Directed Mutagenesis Approaches:

  • Generate CLIC3 mutants with alterations to critical cysteine residues

  • Evaluate functional consequences on oxidoreductase activity

  • Assess impact on protein-protein interactions

  • Determine effects on cellular phenotypes when expressed

Research has demonstrated that CLIC3 functions as a glutathione-dependent oxidoreductase, with the capacity to reduce specific cysteines in proteins like TGM2. For example, cysteine 505 in TGM2 was found to be fivefold more reduced when incubated with enzymatically active CLIC3 compared to the inactive C22A mutant .

What are the recommended approaches for analyzing CLIC3 expression and localization in tissue samples?

For effective analysis of CLIC3 expression and localization in tissue samples:

• Immunohistochemistry Protocol:

  • Perform antigen retrieval on formalin-fixed paraffin-embedded sections

  • Block with appropriate serum to minimize non-specific binding

  • Incubate with validated anti-CLIC3 antibodies

  • Use detection systems appropriate for the tissue type

  • Quantify expression using histoscoring methods (0-300 scale)

• Tissue Sample Analysis:

  • Compare CLIC3 expression between tumor and adjacent normal tissue

  • Assess both stromal and cancer cell compartments separately

  • Co-stain with markers such as αSMA and TGM2 to identify stromal localization

  • Correlate expression patterns with clinical parameters

• Single-Cell Analysis:

  • Employ laser capture microdissection to isolate specific cell populations

  • Perform RT-qPCR or RNA-seq on isolated cells

  • Integrate with spatial transcriptomics data when available

  • Validate findings with in situ hybridization techniques

Studies have demonstrated that CLIC3 localization varies by cancer type, with expression detectable in both cancer cells and stromal compartments. In ovarian tumors, for example, 90% showed positive CLIC3 staining in the stroma, while this was observed in only 20% of breast cancers and rarely in pancreatic cancers .

How does CLIC3 contribute to cancer progression, and what experimental models best demonstrate this?

CLIC3 demonstrates complex roles in cancer progression that vary by cancer type:

• Cancer-Specific Functions:

  • In bladder cancer: Promotes cell proliferation by reducing p21 expression

  • In gastric cancer: Functions as a channel in plasma membrane; decreased expression correlates with unfavorable prognosis

  • In breast cancer: Secreted by cancer-associated fibroblasts to promote invasion

  • In ovarian cancer: High expression correlates with poor survival

• Experimental Models:

  • In vitro cellular models:

    • Knockdown/overexpression studies in cancer cell lines

    • Co-culture systems with cancer-associated fibroblasts

    • 3D matrix invasion assays with recombinant CLIC3

  • Ex vivo models:

    • Mouse aortic ring sprouting assays with VEGF and rCLIC3

    • Fibrin gel endothelial cell sprouting assays

  • In vivo models:

    • Xenograft models with manipulated CLIC3 expression

    • Orthotopic tumor models to assess invasion and metastasis

• Mechanistic Pathways:

  • CLIC3 interacts with NAT10 to inhibit N4-acetylcytidine modification

  • Extracellular CLIC3 functions through TGM2-dependent matrix stiffening

  • TGF-β signaling pathway may be implicated in some contexts

For example, in vitro studies demonstrated that recombinant CLIC3 promoted the extension of invasive pseudopods from MDA-MB-231 breast cancer cells in a dose-dependent manner. Additionally, CLIC3 knockdown in cancer-associated fibroblasts reduced their ability to promote endothelial cell sprouting in 3D fibrin gels, indicating CLIC3's role in tumor angiogenesis .

What are the most significant contradictions or knowledge gaps in current CLIC3 research?

Several key contradictions and knowledge gaps exist in the current understanding of CLIC3:

• Contradictory Prognostic Associations:

  • In gastric cancer: Lower CLIC3 expression correlates with poorer prognosis

  • In bladder cancer: Higher CLIC3 expression correlates with poorer prognosis

  This contradiction suggests context-dependent functions that require further investigation through comparative molecular analyses of CLIC3 signaling networks across different cancer types .

• Unclear Relationship Between Channel and Oxidoreductase Functions:

  • How CLIC3 transitions between soluble and membrane-inserted forms remains poorly understood

  • The relationship between ion channel activity and redox function in physiological contexts needs clarification

  • Whether these functions operate independently or synergistically remains to be determined

• Limited Information on Regulation of CLIC3:

  • Transcriptional control mechanisms remain largely unexplored

  • Post-translational modifications affecting function are poorly characterized

  • Factors controlling secretion versus intracellular retention are not well defined

• Technical Challenges:

  • No recordings of CLIC3 activity in native environments

  • Variable conductance (3-400 pS) reported across experimental systems

  • Limited availability of specific inhibitors for functional studies

Addressing these knowledge gaps requires integrative approaches combining structural biology, electrophysiology, and advanced imaging techniques in physiologically relevant model systems .

How can CLIC3 be effectively targeted or modulated for potential therapeutic applications?

Based on current understanding, several strategies for therapeutic targeting of CLIC3 can be proposed:

• Small Molecule Inhibitors:

  • Target the glutathione-binding site to inhibit oxidoreductase activity

  • Develop compounds that prevent membrane insertion to block channel function

  • Design allosteric modulators that stabilize the soluble form

• Biologics and Antibody Approaches:

  • Develop neutralizing antibodies against secreted extracellular CLIC3

  • Create recombinant proteins that compete with CLIC3 for TGM2 binding

  • Engineer decoy receptors to sequester secreted CLIC3

• Genetic Modulation Strategies:

  • siRNA or antisense oligonucleotides for transient knockdown

  • CRISPR-Cas9 gene editing for permanent alteration

  • Epigenetic modifiers targeting CLIC3 promoter regions

• Combination Approaches:

  • Co-targeting CLIC3 and TGM2 pathways simultaneously

  • Combining CLIC3 inhibition with conventional chemotherapeutics

  • Integrating CLIC3 targeting with immune checkpoint inhibitors

For bladder cancer specifically, CLIC3 represents a promising therapeutic target given its role in promoting cancer cell proliferation through interactions with NAT10 and subsequent downregulation of p21 mRNA stability .

What computational approaches are most effective for predicting CLIC3 functions and interactions?

Advanced computational methods for studying CLIC3 include:

These computational approaches have been successfully applied in studies of CLIC3 in cancers like bladder cancer and acute myeloid leukemia, providing insights into pathways and potential therapeutic targets .

How do post-translational modifications affect CLIC3 function and localization?

While limited information exists on post-translational modifications (PTMs) of CLIC3 specifically, researchers can investigate this critical area through:

• Mass Spectrometry-Based Approaches:

  • Perform phosphoproteomics to identify phosphorylation sites

  • Use redox proteomics to characterize modifications of cysteine residues

  • Employ glycoproteomics to detect potential glycosylation events

  • Analyze ubiquitination and SUMOylation patterns that may affect stability

• Site-Directed Mutagenesis Studies:

  • Generate mutants of predicted PTM sites (e.g., phosphomimetic substitutions)

  • Assess effects on:

    • Subcellular localization

    • Membrane insertion capability

    • Chloride channel activity

    • Glutathione-dependent oxidoreductase function

    • Protein-protein interactions

• Cell-Based Assays for PTM Function:

  • Expose cells to phosphatase inhibitors or kinase activators to alter phosphorylation status

  • Manipulate cellular redox environment to modify redox-sensitive residues

  • Use inhibitors of specific post-translational modification enzymes

  • Monitor changes in CLIC3 localization and function under these conditions

Based on current understanding of the CLIC family, cysteine modifications are likely crucial for CLIC3 function, particularly for the transition between soluble and membrane-associated forms .

What is the significance of CLIC3 in non-cancer pathological conditions?

While much research has focused on CLIC3 in cancer, its potential roles in other pathological conditions warrant investigation:

• Cardiovascular Disorders:

  • CLIC3's ability to activate TGM2 and promote blood vessel growth suggests potential roles in:

    • Atherosclerosis progression

    • Cardiac remodeling after injury

    • Vascular complications in metabolic disorders

• Inflammatory and Fibrotic Conditions:

  • Given CLIC3's involvement in matrix stiffening and tissue remodeling:

    • Pulmonary fibrosis

    • Liver cirrhosis

    • Chronic kidney disease

    • Rheumatoid arthritis

• Neurodegenerative Diseases:

  • Considering the importance of chloride channels and redox regulation in neuronal function:

    • Potential roles in oxidative stress responses

    • Neuroinflammatory processes

    • Protein aggregation disorders

• Developmental Processes:

  • CLIC3's involvement in cellular growth control suggests potential roles in:

    • Embryonic development

    • Tissue morphogenesis

    • Stem cell differentiation

Research methodologies to explore these areas should include:

• Tissue-specific knockout or transgenic mouse models

• Single-cell transcriptomics of affected tissues

• Proteomics analysis of disease-relevant compartments

• Functional studies in organ-specific primary cell cultures

While direct evidence in these areas is currently limited, the multifunctional nature of CLIC3 suggests its potential importance beyond cancer biology .

What are common challenges in CLIC3 functional assays and how can they be overcome?

Researchers may encounter several technical challenges when working with CLIC3:

• Protein Stability Issues:

  • Problem: Recombinant CLIC3 may lose activity during storage or experimental manipulation

  • Solution: Add reducing agents (1-5 mM DTT or 2-10 mM GSH) to all buffers; store at -80°C in single-use aliquots; avoid repeated freeze-thaw cycles

• Variability in Channel Activity Recordings:

  • Problem: Inconsistent channel conductance measurements across experiments

  • Solution: Standardize membrane composition in bilayer experiments; control pH (optimally 7.2-7.4) and redox environment; use site-directed mutagenesis to identify critical residues for channel function

• Difficulties in Detecting Secreted CLIC3:

  • Problem: Low abundance of secreted CLIC3 in culture supernatants

  • Solution: Concentrate conditioned media using ultrafiltration; develop sensitive ELISA assays; use mass spectrometry with targeted MRM approaches for quantification

• Inconsistent Results in Cell-Based Assays:

  • Problem: Variable effects of CLIC3 manipulation across cell lines

  • Solution: Carefully characterize baseline CLIC3 expression in each cell line; use multiple siRNAs or shRNAs for knockdown; validate overexpression constructs for proper localization and function

• Challenges in Measuring Oxidoreductase Activity:

  • Problem: Interference from cellular components in activity assays

  • Solution: Use purified recombinant proteins for in vitro assays; include appropriate controls for non-enzymatic reactions; optimize buffer conditions for maximal activity

By addressing these technical challenges, researchers can generate more reliable and reproducible data on CLIC3 functions .

How can researchers effectively validate CLIC3 knockdown or overexpression in experimental models?

Proper validation of CLIC3 manipulation is crucial for experimental rigor:

• mRNA Level Validation:

  • Perform RT-qPCR using primers spanning different exons

  • Include housekeeping genes appropriate for the cell type/tissue

  • Present data as fold change relative to control

  • Validate using at least two primer sets targeting different regions

• Protein Level Validation:

  • Western blotting with validated antibodies

  • Immunofluorescence to assess subcellular localization

  • Flow cytometry for quantitative analysis in cell populations

  • ELISA for secreted CLIC3 in conditioned media

• Functional Validation:

  • Electrophysiology to confirm changes in chloride channel activity

  • Glutathione-dependent oxidoreductase activity assays

  • Matrix stiffness measurements to assess extracellular effects

  • Cell phenotype assays (proliferation, invasion) relevant to CLIC3 function

• Rescue Experiments:

  • Re-express CLIC3 in knockdown models to restore function

  • Use mutant variants to identify critical domains/residues

  • Add recombinant CLIC3 protein to complement cellular knockout

  • Employ inducible expression systems for temporal control

For example, in studies of bladder cancer, CLIC3 knockdown was validated by showing reduced protein expression via Western blot, followed by functional validation through cell proliferation assays that demonstrated attenuated growth in CLIC3-depleted cells .

What are the optimal methods for analyzing CLIC3 expression in patient samples for prognostic evaluation?

For clinical assessment of CLIC3 as a prognostic marker:

• Tissue Microarray Analysis:

  • Process formalin-fixed paraffin-embedded tissue samples

  • Stain with validated anti-CLIC3 antibodies

  • Use histoscoring method (0-300 scale) to quantify expression

  • Evaluate both cancer cells and stromal compartments separately

  • Correlate with clinical parameters and survival data

• Gene Expression Analysis:

  • Extract RNA from fresh or archived tissue samples

  • Perform RT-qPCR for targeted CLIC3 expression analysis

  • Consider RNAseq for broader pathway analysis

  • Establish cutoff values for high versus low expression using:

    • Median expression

    • ROC curve analysis

    • X-tile software

• Statistical Analysis for Prognostic Evaluation:

  • Use Kaplan-Meier method with log-rank test for survival analysis

  • Perform Cox univariate and multivariate analyses

  • Calculate hazard ratios with 95% confidence intervals

  • Consider P < 0.05 as statistically significant

  • For gene set analysis, use FDR < 0.25 as significance threshold

• Data Presentation:

  • Present survival curves with clear indication of patient numbers

  • Include tables of patient demographics and clinical characteristics

  • Stratify analysis by relevant clinical parameters

  • Report concordance statistics (C-index) for prognostic models

These approaches have been successfully implemented in studies of CLIC3 in various cancers, revealing its prognostic significance. For example, in bladder cancer, CLIC3 overexpression was found to be negatively correlated with patient survival, suggesting its potential as a prognostic biomarker .