Figure 1.
During the lag phase, the aggregation process is reversible and aggregate size remains low. Once a critical size of aggregate is reached, the process is irreversible and the aggregation process is accelerated (exponential growth phase). The pool of aggregatable protein is eventually depleted and the curve reaches a plateau.
Figure 2.
Simulation results for normal conditions.
This shows how some of the species in the model vary over time in a typical simulation run. A Generic pool of protein. NatP = Native protein; TotalMisP = unbound misfolded protein + all bound forms of misfolded protein; Total protein = NatP + TotalMisP. B Ubiquitin pools. Ub conjugates includes all complexes containing ubiquitin not just ubiquitinated proteins (e.g. Ub-UCHL1 complex is included in this pool). C Aggregated protein. SeqAggP = aggregates sequestered into inclusion bodies; AggP = small unbound aggregates (of all types); AggP_Proteasome = small aggregates bound to the proteasome. D α-synuclein levels: asyn = unbound α-synuclein; asyn_dam = total pool of damaged α-synuclein (except any that is present in inclusions); total asyn = total pool of α-synuclein. E Pool of unbound proteasomes. F UCHL1 pools: Free UCHL1 = unbound UCHL1; Total UCHL1 = total pool of UCHL1 (except any that is present in inclusions); UCHL1 substrate = total level of the UCHL1 substrate (either bound or unbound).
Figure 3.
UCH-L1 inclusion formation in COS7 cells over-expressing wild type UCH-L1.
Cells were transfected with UCH-L1-HA for a total of 28 h. 5 µM MG132 was added for the times indicated prior to the end of the experiment. A Typical example of cells overexpressing UCHL1-HA which contain inclusions. Images immunostained with monoclonal HA (green) and counterstained with DAPI to visualise nuclei (blue). Arrows indicate the position of inclusions within the cell. B Time course for UCH-L1 inclusion formation. The presence of UCH-L1 was assessed by immunofluorescence using mouse monoclonal anti-HA antibodies. Bars represent the percentage of cells containing inclusions. Error bars indicate the standard error from the mean of the experiments. Asterisk indicates a significant difference between the percentage of untreated cells versus the percentage of treated cells. *p<0.001. no TX = no treatment. C Comparison of experimental data and simulation results for the percentage of cells containing inclusions in each experiment. Error bars for simulated data represent the standard error for a percentage (s.e = , where p is the percentage and n is the number of simulations).
Table 1.
Time course of inclusion formation from stochastic model simulations.
Table 2.
Time course of inclusion formation from deterministic model simulations.
Figure 4.
Overexpression of UCH-L1 (3x baseline) with PI at 20 hours (shown by vertical dashed line).
A Generic pool of protein. NatP = Native protein; TotalMisP = unbound misfolded protein + all bound forms of misfolded protein; Total protein = NatP + TotalMisP. B Ubiquitin pools. Ub conjugates includes all complexes containing ubiquitin not just ubiquitinated proteins (e.g. Ub-UCHL1 complex is included in this pool). C Aggregated protein. SeqAggP = aggregates sequestered into inclusion bodies; AggP = small unbound aggregates (of all types); AggP_Proteasome = small aggregates bound to the proteasome. D α-synuclein levels: asyn = unbound α-synuclein; asyn_dam = total pool of damaged α-synuclein (except any that is present in inclusions); total asyn = total pool of α-synuclein. E Pool of unbound proteasomes. F UCHL1 pools: Free UCHL1 = unbound UCHL1; Total UCHL1 = total pool of UCHL1 (except any that is present in inclusions); UCHL1 substrate = total level of the UCHL1 substrate (either bound or unbound).
Figure 5.
A–F Six simulation runs showing variability in aggregation kinetics when UCH-L1 is overexpressed (x3) and proteasome is inhibited at time t = 20 h (shown by vertical dashed line).
SeqAggP = aggregates sequestered into inclusion bodies; AggP = small unbound aggregates (of all types); AggP_Proteasome = small aggregates bound to the proteasome.
Figure 6.
UCH-L1 inclusion formation in COS7 cells over-expressing mutant forms of UCH-L1.
A Cells were transfected with wild type or mutant UCH-L1 HA tagged plasmids for a total of 28 h. 5 µM MG132 was added for the times indicated prior to the end of the experiment. Time course for formation. The presence of UCH-L1 was assessed by immunofluorescence using polyclonal UCH-L1 antibodies. Bars represent the percentage of cells containing inclusions. Error bars indicate the standard error from the mean of the experiments. Asterisk indicates a significant difference between the percentage of untreated cells versus the percentage of treated cells. **p<0.001. no TX = no treatment. B Percentage of simulated cells with inclusions for wild-type or mutant UCHL-L1. Error bars indicate standard error for a percentage (s.e = , where p is the percentage and n is the number of simulations).
Figure 7.
The involvement of UCH-L1 in exacerbating any disturbances in protein homeostasis: A under normal conditions, damaged proteins are cleared by proteasomes or lysosomes and there is no protein aggregation; B when proteasomes and/or lysosomes become inhibited, UCH-L1 levels rise and the pool of damaged UCH-L1 also increases.
This leads to inhibition of chaperone-mediated autophagy and also less degradation of UCH-L1 substrates via the ubiquitin-proteasome system. As a result protein aggregates start to form leading to further proteasome inhibition and increased levels of oxidative stress. The grey scale indicates the abundance of each species with white representing very low levels and dark grey representing high levels. Degradation is represented by the empty set symbol ().