Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion

Cholesterol Promotes Interaction of the Protein CLIC1 with Phospholipid Monolayers at the Air–Water Interface

CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion-channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre-incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1-cholesterol pre-complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels

X‑ray and Neutron Reflectivity Study Shows That CLIC1 Undergoes Cholesterol-Dependent Structural Reorganization in Lipid Monolayers

CLIC1 belongs to the ubiquitous family of chloride intracellular ion channel proteins that are evolutionarily conserved across species. The CLICs are unusual in that they exist mainly as soluble proteins but possess the intriguing property of spontaneous conversion from the soluble to an integral membrane-bound form. This conversion is regulated by the membrane lipid composition, especially by cholesterol, together with external factors such as oxidation and pH. However, the precise physiological mechanism regulating CLIC1 membrane insertion is currently unknown. In this study, X-ray and neutron reflectivity experiments were performed to study the interaction of CLIC1 with different phospholipid monolayers prepared using POPC, POPE, or POPS with and without cholesterol in order to better understand the regulatory role of cholesterol in CLIC1 membrane insertion. Our findings demonstrate for the first time two different structural orientations of CLIC1 within phospholipid monolayers, dependent upon the absence or presence of cholesterol. In phospholipid monolayers devoid of cholesterol, CLIC1 was unable to insert into the lipid acyl chain region. However, in the presence of cholesterol, CLIC1 showed significant insertion within the phospholipid acyl chains occupying an area per protein molecule of 6−7 nm² with a total CLIC1 thickness ranging from ∼50 to 56 Å across the entire monolayer. Our data strongly suggests that cholesterol not only facilitates the initial docking or binding of CLIC1 to the membrane but also promotes deeper penetration of CLIC1 into the hydrophobic tails of the lipid monolayer

Evidence of the Key Role of H₃O⁺ in Phospholipid Membrane Morphology

This study explains the importance of the phosphate moiety and H₃O⁺ in controlling the ionic flux through phospholipid membranes. We show that despite an increase in the H₃O⁺ concentration when the pH is decreased, the level of ionic conduction through phospholipid bilayers is reduced. By modifying the lipid structure, we show the dominant determinant of membrane conduction is the hydrogen bonding between the phosphate oxygens on adjacent phospholipids. The modulation of conduction with pH is proposed to arise from the varying H₃O⁺ concentrations altering the molecular area per lipid and modifying the geometry of conductive defects already present in the membrane. Given the geometrical constraints that control the lipid phase structure of membranes, these area changes predict that organisms evolving in environments with different pHs will select for different phospholipid chain lengths, as is found for organisms near highly acidic volcanic vents (short chains) or in highly alkaline salt lakes (long chains). The stabilizing effect of the hydration shells around phosphate groups also accounts for the prevalence of phospholipids across biology. Measurement of ion permeation through lipid bilayers was made tractable using sparsely tethered bilayer lipid membranes with swept frequency electrical impedance spectroscopy and ramped dc amperometry. Additional evidence of the effect of a change in pH on lipid packing density is obtained from neutron reflectometry data of tethered membranes containing perdeuterated lipids

Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function

Conserved G-site motif in members of the CLIC family.

<p>Multiple sequence alignment of human proteins: CLIC 1-6, GST-omega and Grx1-3. Highlighted in grey is the glutaredoxin/thioredoxin active site motif (G-site) (Accession numbers: CLIC1 (CAG46868), CLIC2 (CAG03948), CLIC3 (CAG46863.1), CLIC4 (CAG38532), CLIC5 (AAF66928), CLIC6 (NP_444507), GST-omega (AAF73376), Grx-1 (BAAO4769), Grx-2 (AAK83089) and Grx-3 (AAH0528289) obtained from ClustalW.</p

Glutathione-dependant activity of the CLIC proteins.

<p>(<b>A</b>) The reaction mixture contained 2 mM EDTA in 0.1 M Tris-HCl (pH 7.5), 5 uM reduced CLIC1, CLIC2 or CLIC4 (WT) protein, 200 uM NADPH, 750 uM HEDS, 50 nM TrxR and 5 uM Trx-1 (included as a positive control). (<b>B</b>) Insulin disulfide reductase assay to determine catalytic activity of Trx-1 and CLIC1 based on solution turbidity monitored by A_{650 nm} over 30 minutes.</p

Oxidoreductase activity of the CLIC proteins.

<p>Oxidoreductase enzymatic activity was measured using 5 uM of CLIC proteins or HcTrx-5 or Grx-1, 250 uM NADPH, 1 mM HEDS and 50 nM GR. The reaction was initiated by the addition of 1 mM GSH and the absorbance of NADPH was monitored at A_{340 nm}. Reaction conditions: 5 mM potassium phosphate with 1 mM EDTA, pH 7, at 37°C. (<b>A</b>) Activity of CLIC1, CLIC2 and CLIC4 compared to HcTrx-5 and Grx-1 (positive controls). (<b>B</b>) Activity of 5 uM CLIC1 dimer compared to 5 uM CLIC1 monomer. <i>Error bars</i> represent the S.E. of at least three independent measurements.</p

Effect of chloride ion channel inhibitor drugs on the oxidoreductase enzymatic activity of CLIC1.

<p>5 uM of CLIC1 reduced (WT) or HcTrx-5 protein was incubated with 10 uM IAA-94, A9C, DIDS or Saxitoxin for ∼1 hour prior use of the protein in the assay. The enzyme assay mixture contained 250 uM NADPH, 1 mM HEDS, 50 nM GR in 5 mM potassium phosphate buffer with 1 mM EDTA, pH 7, at 37°C. The consumption of NADPH was monitored at A_{340 nm} post addition of 1 mM GSH. <i>Error bars</i> shown represent the S.E. of at least three experimental measurements.</p

Effect of cholesterol on the enzymatic activity of CLIC1.

<p>5 uM of CLIC1 monomer (WT) protein was incubated with 0.4, 0.8 and 1.6 mM cholesterol for ∼1 hour prior to its addition to a reaction mixture of 250 uM NADPH, 1 mM HEDS, 50 nM GR in 5 mM potassium phosphate buffer with 1 mM EDTA, pH 7, at 37°C. The consumption of NADPH was monitored at A_{340 nm} post addition of 1 mM GSH. Control included all the reaction components including 1.6 mM cholesterol, except with no CLIC1 protein. The <i>error bars</i> shown represent the S.E. of at least three experimental measurements.</p