Degree the vertebrate with a 35% sequence identity

Degree of Conservation between CLIC


There are currently six known
chloride intracellular ion channel (CLIC) proteins (CLIC1 – CLIC6) which have
been found in vertebrates, all possessing a high degree of conservation between
them, suggesting that they have evolved through duplication from the same
ancestral protein in an ancient chordate (Litter et al., 2010). This theory is
strengthened, as urochordate Ciona
intestinalis possesses a single CLIC protein which contains a 45% sequence
identity (highly homologous) to the vertebrate paralogues and also have the
characteristic three conserved cysteine residues within the sequence. Related
CLIC proteins have also been found in invertebrates, whose sequences diverge
slightly from the vertebrate with a 35% sequence identity and most contain the
conserved three characteristic cysteines, with the only exception being some
nematodes, where an aspartate takes the place of the active site cysteine
residue (Littler et al., 2010).

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Function of CLIC Proteins


CLIC proteins have only recently been
discovered and are found to be highly conserved throughout the body but
localise specifically in various subcellular compartments such as organelles,
the cortical actin cytoskeleton, the plasma membrane, vesicles and dependent on
the cell type, centrosomes (Fernandez-Salas et al., 2002; Proutski et al.,
2002; Berryman and Goldenring, 2003; Suh et al., 2003). It has been put in to
question their proposed function of forming chloride channels, as it is
believed they can perform alternative cellular processes. Specific biological
processes in which they have been found to be involved in include, keratinocyte
differentiation, apoptosis, receptor trafficking, endothelial vacuole formation
and tubulogenesis (Bohman et al., 2005; Suh et al., 2007; Maeda et al., 2008;
Tung et al., 2009; Ulmasov et al., 2009). CLIC4 has also been linked with
membrane trafficking and / or cytoskeletal trafficking since it is able to
interact with brain dynamin-I in a complex with actin and tubulin (Suginta et
al., 2001). These additional roles of CLIC proteins give more indication why
over or under expression of these proteins is linked to some disease states,
including many neurodegenerative disorders and various cancers (Averaimo et
al., 2010). 


Structure of CLIC Proteins


CLIC proteins are found under reducing conditions in the
cytosol in a soluble globular form, which has a very close structural
similarity to the omega-type glutathione S-transferases (GSTs) (Board et al.,
2000), and are therefore composed of two domains; an N-terminal thioredoxin-like
domain (residues 16 – 105) that binds to glutathione (GSH) and a highly
conserved all ? helical C-terminal domain, of approximately 240 amino acids,
which binds to a second unknown substrate next to GSH to enable them to
conjugate. The second binding site of the xenobiotic compound is less conserved
than the GSH-binding site and is therefore harder to define. The binding site
of GSH closely resembles glutaredoxin and is found highly conserved in all
CLICs, suggesting that the chloride ion channel activity is controlled by
redox-active signalling molecules in vivo.
CLIC1 is able to bind to glutathione covalently in oxidising conditions,
through a mixed disulphide bond with the redox active cysteine (Cys24) present
within the binding site (Harrop et al., 2001). Therefore, in a resting cell,
the soluble globular form of CLIC1 in the cytosol will be free of glutathione
as the Cys24 will be in a reduced state. In CLIC1 the thiol of Cys24,
consisting of the conserved residues Arg29, cis-proline Pro65 and Asp76, is
regarded as a highly reactive thiolate possessing a low pKa as a result of its
positioning of helix 1 in the N-terminus and the basic properties of conserved
Arg29. Hence, it is highly likely that CLIC1 and all other CLIC proteins are
GSH-dependent redox-active proteins (Harrop et al., 2001).


As seen in figure 1, the structure of CLIC4 shows a break in
density between residues 163 and 173, referring to the flexible foot loop, only
found in vertebrate CLIC proteins as GST or invertebrate CLIC proteins do not
possess it.  It is located between helix
5 and helix 6, with the N-terminus thought to be held in position by
interactions with different residues in close proximity to the reactive Cys35
residue of nearby molecules. It also possesses a joint-like mechanism, as it is
able to hinge at residues Pro158 and Arg176 (conserved in all vertebrate CLIC
proteins (Litter et al., 2005)), whereby the guanidium side chain group of
Arg176 enables the generation of a charged hydrogen-bonding network; an
identical structure formation occurs in the CLIC1 protein.


CLIC4 also contains a putative
internal nuclear localisation sequence (NLS) (residues 199 – 206, figure 1), at
helix 6 of the C-terminus, whereby its solvent exposed face consists of mainly
basic lysine residues (Lys199, Lys203 and Lys204) with Arg206 positioning
itself in the opposing direction to the lysine’s in the loop of helix 6
(Littler et al., 2005). The structure of the NLS peptide in CLIC1 is again near
identical to that of CLIC4 with the only exception being that residue Lys199 is
equivalent to Gln188 in CLIC1. The NLS is of high importance to the protein as
it is able to control its nuclear import machinery. This is regulated by helix
6 of the NLS partially unfolding to encourage the binding of targeted
importin/karyopherin family proteins to the NLS peptide, to allow further
conformational changes causing the basic NLS residues to be inserted into their
specific binding pocket (Litter et al., 2005).


CLIC4 is also found in an integral
membrane form and it has been hypothesised that it contains putative
transmembrane (PTM) domain near the N-terminus which runs approximately from
Cys35 to Val57 (Figure 1). The presence of a transmembrane region can be
supported by performing proteinase K treatment of microsomes containing CLIC
proteins, resulting in a 27 kDa reduction in size for CLIC4 protein which
leaves only a 6 kDa fragment (Duncan et al., 1997). Another notable feature within CLIC proteins is
the presence of several cysteine residues, making them susceptible to
intrachain and/or inter-subunit disulphide bond formation, which in some cases
aids in the conformational changes required for integral membrane form. 


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