Engineered disulfide bonds restore chaperone-like function of DJ-1 mutants linked to familial Parkinson's disease

Abstract

Loss-of-function mutations such as L166P, A104T, and M26I in the DJ-1 gene (PARK7) have been linked to autosomal-recessive early onset Parkinson's disease (PD). Cellular and structural studies of the familial mutants suggest that these mutations may destabilize the dimeric structure. To look for common dynamical signatures among the DJ-1 mutants, short MD simulations of up to 1000 ps were conducted to identify the weakest region of the protein (residues 38-70). In an attempt to stabilize the protein, we mutated residue Val 51 to cysteine (V51C) to make a symmetry-related disulfide bridge with the preexisting Cys 53 on the opposite subunit. We found that the introduction of this disulfide linkage stabilized the mutants A104T and M26I against thermal denaturation, improved their ability to scavenge reactive oxygen species (ROS), and restored a chaperone-like function of blocking alpha-synuclein aggregation. The L166P mutant was far too unstable to be rescued by introduction of the V51C mutation. The results presented here point to the possible development of pharmacological chaperones, which may eventually lead to PD therapeutics.

Figure 1

(a) Ribbon representation of the two subunits of DJ-1 showing the positions of the various familial Parkinson’s disease causing mutations and the disulfide linkages (side panel) used in this investigation. Table -1 shows the stereochemical parameters for the disulfide bonds generated as a result of V51C mutation. (b) 4–12% gradient SDS-PAGE analysis of disulfide bond formation in DJ-1^V51C, DJ-1^L166P/V51C, DJ-1^M26I/V51C and DJ-1^V51C/A104T under non-reducing conditions. The lanes are as follows: Lane 1 is molecular weight marker and Lane 2 is DJ-1^WT. Lanes 3–6 are DJ-1^V51C, DJ-1^M26I/V51C, DJ-1^A104T/V51C and DJ-1^L166P/V51C respectively under non-reducing conditions. Lanes 7–9 are DJ-1^V51C, DJ-1^M26I/V51C and DJ-1^A104T/V51C under reducing conditions. (c) Thermal melting curves (fluorescence) for DJ-1^WT and various mutants measured using thermofluor (sypro orange) on a realtime PCR machine. Thermal melting curves for each protein sample was measured from three independent wells (n=3) and the averaged data is shown here. The melting curves indicate that the DJ-1^WT is the most stable with Tm=65±0.5°C. The familial mutants were found to be less stable than WT with DJ-1^M26I (Tm=52±0.5°C) and DJ-1^A104T(Tm=53±0.5°C). The V51C disulfide-linked versions of the mutant DJ-1 were more stable compared to the familial mutants (DJ-1^M26I/V51C Tm=57±0.5°C, DJ-1^A104T/V51C Tm=58±0.5°C)

Figure 2

(a) R.M.S deviation per residue for the Cα atom for DJ-1^WT, the familial Parkinson’s disease mutants and their disulfide versions DJ-1^M26I/V51C and DJ-1^A104T/V51C observed over the course of 1000ps MD simulation. The maximum variability between WT and the various mutants is observed between residues 38 and 70, parts of which coincide with the dimer interface and include the V51 region. Other region of the protein with high mobility includes the solvent exposed helix 6 between residues 127–138. Key residues important in this investigation and indicated by arrows and the dimer interface residues are indicated by DI. Among the mutants DJ-1^A104T and DJ-1^L166P appear to have more perturbation compared to DJ-1^M26I around the 38–70 region. Simulation trajectories of the disulfide bonded counterparts of these mutants are indicated by brown dotted line (DJ-1^A104T/V51C) and pink line (DJ-1^M26I/V51C). (b) Ribbon representation of DJ-1^A104T representing the molecular motions in the various parts of the protein. The per-residue R.M.S variation of figure 2a was converted to B-factors and ramped using the coloring scheme indicated below (blue; R.MS.D = 0.0 Å, red; R.M.S.D = 4.0 Å). Two mobile regions including the dimer interface residues (38–70) and helix 6 (127–138) are indicated by arrows.

Figure 3

(a) Autooxidation of Cys 106 of DJ-1^WT, familial mutants and disulfide bonded (V51C) versions of the familial mutants are compared using a glutathione-peroxidase (PX) assay kit. 100nM of glutathione peroxidase (PX) was used as a positive control in this assay. The disulfide mutant DJ-1^A104T/V51C showed increased ability to autooxidize compared to DJ-1^A104T (n=5, , p<0.0001 Student’s t-test). Similarly, DJ-1^M26I/V51C showed higher glutathione peroxidase activity as compared to DJ-1^M26I (n=5, , p=0.0004 Student’s t-test). (b) Aggregation of α-synuclein in presence of various DJ-1^WT and various mutants. Data from five independent aggregation experiments for α-synuclein incubated various forms of DJ-1 was used for generation of the plot and error bars. α-synuclein aggregates to completion within 48hrs under our assay conditions. The mutants DJ-1^A104T and DJ-1^M26I have no effect on α-synuclein aggregation kinetics. DJ-1^WT delays the aggregation of α-synuclein with the first Thio-T positive aggregates appearing around 80hrs. The V51C disulfide bonded mutants DJ-1^A104T/V51C and DJ-1^A104T/V51C delays the aggregation of α-synuclein with the first Thio-T positive aggregates appearing after 60hrs.

Figure 4

Aggregation of α-synuclein in presence of DJ-1^WT and DJ-1^V51C followed over extended periods of time. DJ-1^WT is effective in blocking aggregation; however the effects lasts only till 80hrs (First thio-T positive aggregates observed after 80hrs). The disulfide bonded DJ-1^V51C can exert its anti-aggregation effects for longer periods of time beyond 80hrs. In this experiment, the aggregation kinetics was followed up to 196hrs during which no aggregation of α-synuclein was observed. Data from five independent aggregation assays were used to generate the plot and error bars in this study (b) Electron microscopy analysis of mixtures of DJ-1^WT+α-synuclein and DJ-1^V51C+α-synuclein after 80hrs of incubation (representative electron microscopy field shown here at 30000×). The DJ-1^WT+α-synuclein mixture shows both fibrillar aggregates as well as amorphous aggregates. Analysis of pure samples of α-synuclein in the control experiments shows fibrillar aggregates with amyloid like characteristic, while DJ-1^WT shows amorphous aggregates.