12 research outputs found
A Structural Basis for Cellular Uptake of GST-Fold Proteins
It has recently emerged that glutathione transferase enzymes (GSTs) and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C) is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.This work was supported by Grant DP0558315 Australian Research Council (http://www.arc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
A Structural Basis for Cellular Uptake of GST-Fold Proteins
It has recently emerged that glutathione transferase enzymes (GSTs) and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the a-helical C-terminal domain (GST-C) is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the a-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to,2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the ‘GST-fold ’ structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency
Stability studies of GSTM2-2 and GST-C variants measured by circular dichroism (CD) and differential scanning fluorimetry.
<p>(a) Normalised molecular ellipticity at 222 nm as a function of guanidine
HCl concentration (0–5 M) as measured by CD experiments, GST-C
(■), Y160A (▼) and F157A (▲). (b) Differential scanning
fluorimetry profiles of GST-C wildtype (black), GST-C/Y160A (red),
GST-C/F157A (green) and hen egg-white lysozyme control (blue). Melting
temperatures (T<sub>m</sub>) are denoted by vertical broken lines.</p
Effect of substitution mutations within the GSTM2 α-6 helix upon cellular translocation of the C-terminal domain (GST-C).
<p>L-929 cells were treated for 2 hours with 200 nM Oregon Green-labelled
GST-C variants and GST-C wildtype. The mean cellular fluorescence of
each sample was normalised for the degree of fluorescent labeling of
that protein. Data represents the average of four to six independent
experiments ± SEM. Significantly changed translocation efficiency
(as determined by student's paired t-Test; one-tailed) is indicated
by an asterisk (* = P<0.05;
** = P<0.01).</p
Thermodynamic parameters characterising the guanidine-induced unfolding transition of GST-C and the mutants F157A and Y160A.
<p>Measurements were performed by monitoring the molar ellipticity at
222 nm (CD) using guanidine HCl (0–5 M) as a denaturant.</p
Ribbon structure model of the C-terminal domain of GSTM2, taken from the crystal structure of the full-length protein – PDB file 1XW5.
<p>The α-6 helix is highlighted in bold and is surrounded by other
α-helical elements of GST-C. Residues mutated to probe the role of
the α-6 helix in cell translocation are displayed and labeled.</p
Structural and cell translocation analysis for α-helical fragments derived from GST-C.
<p>(a) Schematic diagram outlining α-helical fragment constructs of
GSTM2. Peptides H6 and H7–8 were synthesized. The peptides in bold
denote fragments that have been tested. (b) Comparative cellular uptake
of GSTM2 C-terminal peptides. L-929 cells were incubated with 200 nM
OG-labelled peptides, GSTM2 protein (X), GST-C (◆) and H4–7
(■), H7–8 (●), H5–8 (▲) fragments of GST-C
for the indicated time periods and intracellular fluorescence measured
by flow cytometry. The mean cell fluorescence of each sample was
normalised for the degree of fluorescent labeling of that protein. Data
represents the average of triplicate measurements from three independent
experiments ± SEM. (c) Circular dichroism spectra for the
full-length GSTM2 protein (dark blue) versus GST-C (green) and
H4–7 (cyan), H7–8 (orange), H5–8 (pink) fragments of
GST-C. All proteins/peptides were measured at 4–5 µM
concentration in 10 mM sodium phosphate buffer pH 7.2.</p
GST-C and GSTM2 proteins show comparable localisation and endocytosis inhibition profiles in L-929 cells.
<p>(a) L-929 cells were incubated for 1 hr with 200 nM GSTM2-OG (A) and 200
nM GST-C-OG (B), washed and then observed by confocal microscopy. The
fluorescence from both GSTM2 and GST-C is in a punctate pattern in the
cytoplasm, whilst minimal cell fluorescence is observed from BSA. (b)
Impact of pathway-specific endocytosis inhibitors on GST-C uptake. L-929
cells were incubated for 2 hrs with 200 nM GST-C alongside GSTM2-OG,
GSTZ1-OG and Sj.GST-OG after inhibition of endocytosis pathways by 8
µg/mL chlorpromazine (white), 5 mM amiloride (light grey), or 10
µg/mL filipin (dark grey). The impact of inhibitors on GST
internalisation was measured quantitatively by flow cytometry. Data is
presented as the percentage intracellular fluorescence in treated cells
compared to intracellular fluorescence in the absence of inhibitors
(dashed line represents uninhibited control). Error bars represent the
SD of three independent experiments.</p