Figure 1.
Expression studies of AGT variants in CHO cells.
A) Levels of AGT immunoreactive protein as determined in soluble (black bars) and insoluble (grey bars) extracts. The inset shows representative immunodetection experiments of AGT variants (upper image) and the corresponding loading controls (lower image); rAGT: recombinant His-tagged AGT. B) AGT activities in soluble extracts. Data are means±s.d. of 2–5 independent experiments. N.D. not detectable.
Figure 2.
Immunolocalization studies of AGT variants in CHO cells.
WT (upper left panel), LM (lower left panel), p.G170R (upper right panel) and p.F152I (lower right panel). In each panel, the upper row shows mitochondrial immunolocalization (AGT variant, Mitotracker probe and their merge), while lower row shows peroxisomal immunolocalization (AGT variant, PMP70 and their merge).
Table 1.
Functional properties and hydrodynamic diameter of AGT variants.
Figure 3.
Interaction of AGT variants with Hsc70 chaperones in a cell-free system.
Representative autoradiograms of AGT proteins labeled with 35S-Met are shown for several AGT variants (E: total AGT synthesized in extracts; I: AGT immunoprecipitated using anti-Hsc70 antibodies; note that 1 µl of the TnT reaction was loaded in E lanes, while the protein immunoprecipitated from 6 µl TnT lysate was loaded in I lanes). Data in the lower panel are expressed as percentage of immunoprecipitated AGT compared to the total AGT synthesized, and are mean±s.d. from three independent experiments.
Figure 4.
Functional characterization of WT and p.H83R.
A and B) Enzyme activity measurements for WT (A) and p.H83R (B) at different L-Ala concentrations in the presence of 0.25 mM (diamonds), 0.5 mM (down triangles), 1 mM (up triangles) and 2 mM (circles) glyoxylate. Data in panel A are means±s.d from four independent measurements while data in panel B are means from two independent measurements. Lines are best fits for the different glyoxylate concentrations obtained from global fittings using a double-displacement mechanism. C and D) Absorption (C) and circular dichroism (D) spectra for holo-WT (black) and holo-p.H83R (grey) acquired upon incubation for at least 10 min in the absence (continuous lines; PLP bound) or presence (dashed lines; PMP bound) of 500 mM L-alanine.
Figure 5.
Thermal denaturation of AGT variants studied by differential scanning calorimetry (DSC).
A) Representative DSC traces obtained at 3°C/min; Lines are best-fits from a two-state irreversible denaturation model with first-order kinetics [6]; B) Arrhenius plots for the irreversible denaturation of AGT variants, indicating also the extrapolated rate constants at physiological temperature (intercept with the vertical dotted line). Symbols are: WT (circle), LM (triangle) and p.F152I (square). Data for holo proteins are shown as closed symbols and for apo proteins as open symbols.
Table 2.
Thermal denaturation and kinetic stability parameters for AGT enzymes.
Figure 6.
Structure-energetics relationships for thermal denaturion of holo- and apo-AGT enzymes.
A) Temperature dependence of denaturation enthalpies (ΔH) for holo- (closed symbols) and apo-(open symbols) proteins. The linear fit provides the value of ΔCp ( = 10.2±0.6 kcal.mol−1). B) Activation energy (Ea) plotted vs. the Tm for holo- (closed symbols) and apo-(open symbols) AGT enzymes. C and D) changes in activation enthalpic and entropic contributions to AGT kinetic stability as a function of changes in activation free energies for holo-(C) and apo-(D) AGT enzymes. Lines in C and D are meant to guide the eye and have no theoretical meaning.
Figure 7.
Structural modeling of AGT mutations and polymorphisms.
A) Representation of AGT dimer structure. The domain structure of one of the subunits is emphasized using green and blue colors. The AGT mutations found in PH1 patients are labeled and pointed by the arrows while the polymorphisms constituting the minor allele are highlighted in magenta. B) Representation of the structural environment of the Arg197 (left), His83 (middle) and Ala295, Pro319 and Ala368 (right). The mutations have been modeled as thinner sticks.
Figure 8.
Folding and misfolding checkpoints of PH1 causing mutants.
Table 3.
Summary of mutational effects on molecular properties of AGT protein.