Recombinant Human Chloride intracellular channel protein 1 (CLIC1)

What is the basic structural transition mechanism of CLIC1?

CLIC1 undergoes a remarkable conformational shift from a soluble cytoplasmic state to a membrane-bound chloride channel. Recent structural biology approaches combining NMR spectroscopy, SAXS, biophysical methods, and mutagenesis reveal that CLIC1 exists in a conformational ensemble characterized by:

• A compact ground state

• A partially extended state that exposes key membrane-interacting regions

• Structural flexibility within dynamic loop regions and interdomain linkers that facilitate membrane adaptation

The transmembrane (TM) region contains pivotal residues (particularly R29 and W35) that modulate protein dynamics, oligomerization, and insertion efficiency. This structural plasticity is intrinsic to CLIC1's function as an ion channel .

How does CLIC1 interact with lipid bilayers?

Fluorescence spectroscopy demonstrates that CLIC1 interacts directly with lipid bilayers under oxidizing conditions. A fluorescence energy transfer (FRET) approach between CLIC1's single tryptophan residue (Trp35) and dansyl-labeled lipid analogues reveals:

• Strong FRET signal between Trp35 and dansyl-lipid analogues under oxidizing conditions

• FRET distance between Trp35 and the dansyl moiety on the membrane surface of approximately 15 Å

• Evidence supporting an oxidation-driven interaction of CLIC1 with lipid bilayers

• A proposed membrane anchoring role for Trp35

These findings provide direct structural evidence of CLIC1-membrane association and support the current model of oxidation-driven membrane interaction .

What experimental techniques are most effective for studying CLIC1 expression?

For accurate CLIC1 expression analysis, quantitative PCR (qPCR) with carefully validated primers is recommended. When implementing qPCR for CLIC1 analysis:

This methodology has successfully demonstrated a 3-fold increase in CLIC1 expression after 8 and 24 hours of LPS stimulation in THP-1 cells .

How does CLIC1 contribute to cancer progression?

CLIC1 functions as a promoter in multiple malignancies through several mechanisms:

In oral squamous cell carcinoma (OSCC), CLIC1:

• Enhances cell proliferation (increasing cell viability and colony formation)

• Inhibits apoptosis (decreasing apoptosis rates measured by flow cytometry)

• Promotes migration and invasion (demonstrated through wound healing and transwell assays)

• Supports angiogenesis

• Decreases drug susceptibility to cisplatin

Mechanistically, CLIC1 modulates:

• Increased levels of ITGαv, ITGβ1, p-ERK, vimentin, MMP2, and MMP9

• Decreased levels of p-p38, E-cadherin, caspase3, and caspase9

• Activation of MAPK/ERK and MAPK/p38 signaling pathways

These findings demonstrate CLIC1's multifaceted role in promoting cancer progression through effects on proliferation, apoptosis, migration, invasion, and chemosensitivity .

What is the prognostic value of CLIC1 in gliomas?

Multi-omics analysis reveals CLIC1 as a significant prognostic biomarker in gliomas:

At the single-cell level, CLIC1 is expressed ubiquitously across different cell types in glioma samples, including macrophages and astrocytes, suggesting its broad involvement in the tumor microenvironment .

How can CLIC1 be utilized as a biomarker in lung adenocarcinoma (LUAD)?

CLIC1 shows promise as a diagnostic biomarker for distinguishing LUAD from normal tissue:

Additionally, CLIC1 expression can complement clinical parameters (age, sex, smoker status, T/N/M staging) in prognosis evaluation, offering superior performance in some cases for evaluating clinical outcomes in patients with various stages of disease .

How do CLIC1 and CLIC4 regulate endothelial cell signaling pathways?

CLIC1 and CLIC4 play distinct roles in mediating G-protein-coupled receptor (GPCR) signaling in endothelial cells:

Shared Functions:

• Both transiently translocate to the plasma membrane in response to sphingosine-1-phosphate (S1P)

• Both are essential for S1P-induced activation of Rac1 downstream of S1PR1

CLIC1-Specific Functions:

• Only CLIC1 is essential for S1P-induced activation of RhoA downstream of S1PR2 and S1PR3

Key Finding: Rescue experiments demonstrate that CLIC1 and CLIC4 are not functionally interchangeable, suggesting distinct and specific functions in transducing GPCR signaling.

These findings establish CLICs as critical mediators of GPCR signaling pathways associated with vascular development and disease, with important implications for understanding vascular biology .

What molecular factors trigger CLIC1 membrane insertion?

The transition of CLIC1 from soluble to membrane-bound states involves several key molecular triggers:

• Zn²⁺ Binding:

  • Acts as a critical trigger inducing structural rearrangements

  • Increases protein flexibility

  • Promotes oligomerization (dimerization and tetramerization) essential for membrane insertion

• Oxidizing Conditions:

  • Fluorescence studies demonstrate strong CLIC1-lipid interactions under oxidizing conditions

  • Oxidation drives conformational changes that expose membrane-interacting regions

• pH Changes:

  • Identified as an environmental trigger controlling CLIC1's metamorphic transition

• Key Residues:

  • R29 and W35 in the transmembrane region play pivotal roles in:

    • Modulating protein dynamics

    • Facilitating oligomerization

    • Enhancing insertion efficiency

This mechanistic framework explains how CLIC1 transitions to its membrane-bound state through the interplay between conformational dynamics, oligomerization, and metal ion modulation .

How does CLIC1 influence immune response in the tumor microenvironment?

Analysis reveals CLIC1's complex role in tumor immunity:

• Tumors with high CLIC1 expression exhibit significantly elevated levels of immune checkpoints (CD40/CD40LG, PDCD1/PDCD1LG2, CTLA4, CD276, IDO1)

• Expression profiles of high-CLIC1 tumors resemble those of patients responding to PD-1 antibody therapy

• Despite this, high CLIC1 expression is associated with resistance to immune checkpoint blockade (ICB):

  • Higher dysfunction scores

  • Higher IFNG scores

  • Higher TIDE scores

These seemingly contradictory findings suggest that although CLIC1-upregulated tumors have abundant immune cells and highly expressed immune checkpoints, they develop mechanisms of resistance to immunotherapy .

What are the optimal techniques for studying CLIC1 structural transitions?

An integrated structural biology approach is recommended for comprehensive analysis of CLIC1 structural transitions:

This multi-technique approach has successfully elucidated the dynamic landscape underpinning CLIC1's remarkable functional versatility .

How can single-cell analysis enhance our understanding of CLIC1 in disease contexts?

Single-cell RNA sequencing (scRNA-seq) provides valuable insights into CLIC1's role in heterogeneous tissues:

Recommended Analysis Pipeline:

• Quality control: Remove cells with >10% mitochondrial UMI counts

• Analysis implementation through Seurat package

• Selection of top 2,000 highly variable genes

• Cell type clustering and identification based on known markers from CellMarker database

• Functional role investigation through GSVA R package

• Cell-cell communication analysis via CellChat R package

• Cellular differentiation assessment using CytoTRACE algorithm

• Pseudotime analysis with monocle2 R package to determine differentiation direction

This approach has revealed CLIC1 expression across multiple cell types in glioma specimens, including macrophages and astrocytes, providing insights into its function within the tumor microenvironment .

What approaches are most effective for evaluating CLIC1 as a potential therapeutic target?

A comprehensive evaluation of CLIC1's therapeutic potential should include:

This multi-faceted approach has successfully identified CLIC1 as a therapeutic vulnerability in gliomas and potentially other cancers .

What are the key unresolved questions about CLIC1 channel formation?

Despite significant advances, several critical aspects of CLIC1 channel formation remain unexplored:

• High-Resolution Membrane-Bound Structure:

  • No high-resolution structural data is currently available for CLIC1's integral membrane state

  • Understanding how CLIC1 unfolds and refolds across the bilayer to form a functional ion channel remains a challenge

• Oligomerization Mechanism:

  • While Zn²⁺-induced dimerization and tetramerization have been identified as key steps preceding insertion

  • The precise arrangement and stoichiometry of CLIC1 oligomers in functional channels remain unclear

• Regulation of Channel Activity:

  • Beyond pH and oxidation, additional physiological regulators of CLIC1 channel function require investigation

  • The interplay between CLIC1 and other ion channels or transporters needs clarification

• Tissue-Specific Functions:

  • How CLIC1 function varies across different tissues and cell types

  • The role of post-translational modifications in regulating tissue-specific activities

Addressing these questions will require innovative approaches combining structural biology, electrophysiology, and advanced imaging techniques .

How might targeted CLIC1 modulation be developed for therapeutic applications?

Based on current understanding, several promising therapeutic strategies targeting CLIC1 could be developed:

• Small Molecule Inhibitors:

  • Design compounds targeting the critical transmembrane region, particularly residues R29 and W35

  • Develop modulators that prevent Zn²⁺-induced oligomerization

• Peptide-Based Approaches:

  • Create peptides mimicking key structural elements that interfere with membrane insertion

  • Design competitive inhibitors of CLIC1-protein interactions in signaling pathways

• Combination Therapies:

  • In cancer contexts, combine CLIC1 inhibition with immune checkpoint inhibitors to overcome resistance

  • Target both CLIC1 and downstream effectors in the MAPK/ERK and MAPK/p38 pathways

• Gene Therapy Approaches:

  • Develop RNA interference or CRISPR-based strategies for tissue-specific CLIC1 modulation

  • Create conditional knockdown systems for research applications

These therapeutic approaches hold particular promise for conditions where CLIC1 is implicated, including various malignancies and cardiovascular diseases .

What are the technical challenges in recombinant CLIC1 production for structural studies?

Producing high-quality recombinant CLIC1 for structural studies presents several challenges:

• Protein Stability:

  • CLIC1's metamorphic nature makes it prone to conformational heterogeneity

  • Conditions must be carefully optimized to maintain the desired conformational state

• Oligomerization Control:

  • Preventing unwanted oligomerization during purification

  • Developing methods to isolate specific oligomeric states for comparative studies

• Membrane-Bound State Preparation:

  • Creating stable preparations of membrane-inserted CLIC1 for structural analysis

  • Designing constructs that facilitate crystallization or cryo-EM studies of the membrane form

• Functional Validation:

  • Ensuring that recombinant CLIC1 retains native channel-forming abilities

  • Developing reliable assays to measure channel activity of purified protein