In 2001, Dr. Kopito’s lab achieved a breakthrough in HD research. Prior to this breakthrough, researchers knew that HD was associated with clumps, or aggregates, of huntingtin protein in nerve cells. (To learn more about huntingtin protein aggregation, click here.) Researchers, however, were not sure if the aggregates actually caused HD or were simply a byproduct of the disease. Kopito’s work suggests that the protein aggregates directly affect a critical cellular mechanism, the ubiquitin-proteasome system (UPS), ultimately resulting in cell death. This breakthrough implies that the aggregates play a causal role in HD.
Normally, proteasomes (a set of cellular enzymes) hunt down and destroy misfolded, unassembled, or damaged proteins that have been tagged by ubiquitin for destruction. Since as many as 80% of cellular proteins fold incorrectly, the UPS is crucial for destroying these misshapen proteins before they cause damage to the cell. (To learn more about the UPS, click here.) But in the case of HD, the UPS fails to do its job. Even though ubiquitin tags huntingtin aggregates as proteins that need to be destroyed, the proteasomes do not seem to destroy them. Therefore, the huntingtin aggregates continue to grow freely. Knowing this process, Kopito sought to investigate whether or not a malfunction of the proteasome itself enables this protein accumulation to occur.
Kopito and Stanford graduate student Neil F. Bence designed an innovative experiment to uncover the relationship between the huntingtin aggregates and the UPS. To test whether or not the UPS is malfunctioning, they engineered a molecule that could detect proteasome activity in a cell. Their detector was an altered form of green fluorescent protein (GFP), a molecule that emits a green fluorescence when placed under a special light. Because altered GFP proteins are rapidly destroyed by functional proteasomes, the level of green fluorescence in a cell could be used to measure functional proteasome activity – with a brightly glowing green cell indicating dysfunctional proteasomes and a non-glowing cell indicating functional proteasomes.
Kopito’s lab then inserted a non-HD allele of the Huntington gene and the altered GFPs into human embryonic kidney cells. Because the embryonic kidney cell was in all other ways normal, and the non-HD allele produced normal huntingtin protein, it was predicted that the cell’s proteasomes would destroy the altered GFP’s, and the cell would not glow. The results were consistent with that prediction. The researchers then predicted that when an HD allele was inserted into the kidney cells, huntingtin protein aggregates would build up, and the proteasomes would fail to destroy the altered GFPs, causing the cell to glow. The result was right on target, with the cells glowing brightly in a matter of hours. (See Figure 1.)
Kopito and Bence also performed the experiment with a different protein, a form of cystic fibrosis membrane conductance regulator protein (CFTR) that has no relation to the huntingtin protein, other than the tendency to also misfold and aggregate. They did this to prove that the UPS breakdown was not specific to huntingtin aggregates, but could be caused by any protein aggregation. The results were identical – the cells expressing the disease-causing CFTR glowed green, indicating a failure in the UPS. Protein aggregation was also shown to lead to the accumulation of ubiquitin conjugates within the cell, as well as stopping the cell cycle process, further suggesting that protein aggregation inhibited the UPS.
Kopito’s research indicates that protein aggregation initiates a vicious cycle: First, the disease produces defective proteins that clump together. As they accumulate, the aggregates put stress on the UPS and interfere with its function, which in turn puts more stress on the UPS and results in the formation of more aggregates that further impair the proteasome. (See Figure 2.) Kopito suggests that the slow buildup of aggregates could explain the late onset of many neurodegenerative diseases, including HD.
While protein aggregates appear to halt the UPS, how they impair the UPS is still unclear. Kopito suggests that the aggregates might get “stuck” in the UPS when the proteasomes attempt to degrade them. When functioning correctly, proteasomes “suck” abnormal proteins through a small opening in their system, much like someone slurping a strand of spaghetti. A “knot” or clump in the “spaghetti” would prevent the strand from passing through. The proteasome might not let go of its prey, however, and might therefore be clogged and out of commission. Kopito’s lab now plans to conduct an experiment to see if this is indeed the case.
Bence, NF; Sampat, RM; Kopito, RR. “Impairment of the Ubiquitin-Proteasome System by protein aggregation.” Science 2001, 292: 1552-1555. A highly technical scientific report describing Kopito’s discovery of the UPS impairment by protein aggregation. In the same magazine, on page 1467, there is a summary that is easier to read.
Shwartz, M. “Scientists close in on cause of neurodegenerative diseases.” Stanford Report, 30 May 2001, 33 (31): 1, 5. A less technical summary on Kopito’s research and the scientific report above.
Last Modified: 05/22/2009
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In 2001, Dr. Kopito’s lab achieved a breakthrough in HD research. Prior to this breakthrough, researchers knew that HD was associated with clumps, or aggregates, of 
