May 25, 2001 Stanford researchers have found an answer to a long-standing mystery surrounding Huntington’s, Alzheimer’s, Parkinson’s and other neurodegenerative diseases.
Their discovery, published in the May 25 issue of the journal Science, focuses on one of the telltale signs of neurodegenerative illness: the mysterious buildup of defective proteins in and around nerve cells.
Healthy cells have the ability to break down and eliminate unwanted proteins. But in neurodegenerative diseases, abnormal proteins clump together to form clusters – called aggregates – that interfere with the cell’s normal functions.
“It’s been known for years that most neurodegenerative diseases are associated with protein aggregates,” says Ron R. Kopito, professor of biological sciences and co-author of the study, “but no one had a clue as to the exact relationship. Do aggregates cause the disease, or are they the result of the disease?”
To find out, Kopito and graduate student Neil F. Bence designed a laboratory experiment to assess the impact of protein aggregates on the inner workings of a cell.
Their specific target was the proteasome – a barrel-shaped enzyme that Kopito calls the master controller of the cell.
“The proteasome is like a salami slicer that cuts protein molecules into little bits,” he says. “It gets rid of abnormal proteins, and it breaks down and recycles regulatory proteins no longer needed by the cell.”
Human cells contain thousands of proteins, each with a unique three-dimensional shape determined by specific genes. But random genetic errors and mutations may cause proteins to fold into the wrong 3-D configuration -– often with devastating results.
One example is huntingtin – a protein found in healthy nerve cells. A slight genetic mutation may cause huntingtin proteins to fold incorrectly and accumulate inside the nerve. Defective huntingtin aggregates are common in patients with Huntington’s disease.
Proteasomes are supposed to slice misfolded proteins into harmless pieces before they have a chance to form aggregates, but how does a proteasome recognize an abnormal protein?
The answer: A tiny molecule called ubiquitin latches onto the damaged protein and carries it to the proteasome, where the protein is sliced and diced.
But in some diseases, the ubiquitin-proteasome system breaks down. Huntingtin aggregates, for example, contain thousands of misfolded proteins with ubiquitin flags attached to them. So why doesn’t the proteasome recognize and destroy these ubiquitin-labeled proteins?
“People have speculated that the reason these proteins accumulate is because the proteasome isn’t functioning properly. In our study, we put that to the test,”Kopito says.
In their lab experiment, Kopito and Bence used human embryonic kidney cells instead of neurons.
To observe proteasome activity in the kidney cells, the researchers devised a genetic tool that causes specific cells to change color. Here’s how it worked: They took advantage of a molecule called green fluorescent protein (GFP), which emits a green fluorescence when placed under a special light.
Although GFP is a very stable molecule, Bence and his co-workers engineered a mutation in the GFP gene that produces unstable proteins, which break down rapidly inside the proteasome. Mutant GFP genes were inserted into the kidney cells, producing unstable proteins that glowed green – but only when they remained intact. When a GFP molecule was chopped into fragments by a proteasome, the green fluorescence did not appear.
“The level of green fluorescence indicated how efficiently the proteasome was breaking down proteins,” says Bence, lead author of the Science study. “If the cell glowed bright green, then we knew the proteasome was not functioning.”
The researchers wanted to see what would happen to unstable GFPs if normal huntingtin genes were inserted into the kidney cells. The result was clear: The cells did not change color – a strong indication that the proteasome enzymes were doing their job slicing up GFP and other unwanted proteins.
But when Bence and Kopito inserted a mutant huntingin gene, the cells turned bright green in a matter of hours. The change in color was accompanied by a buildup of defective protein aggregates inside the cells.
They tried the same experiment using genes that produce mutant forms of CFTR – a protein that has been linked to the disease cystic fibrosis. The results were the same: Cells containing mutant CFTR proteins also formed aggregates and became brightly fluorescent.
“The key finding in our study is that the function of a proteasome can be impaired by the presence of protein aggregates,” Kopito says. “These results provide the first well-documented linkage between protein aggregation and a critical cellular function. If the proteasome isn’t working properly, itís unable to perform its regulatory function, which is very bad for the cell.”
Cause or effect?
Returning to the original question, do aggregates cause diseases, such as Huntington’s and cystic fibrosis, or are they a consequence of disease?
“Our study shows that aggregates themselves can affect the proteasome and cause toxicity,” concludes Bence.
Itís a vicious circle, he points out: “The disease produces defective proteins that clump together into aggregates. Then the aggregates build up and interfere with proteasome function, which results in the production of more aggregates that further impair the proteasome.”
The relatively slow buildup of aggregates in and around nerve cells could explain the latency of many neurodegenerative diseases, adds Kopito.
“The effects of aggregate formation can remain silent for a long time until aggregates build up to the point where onset of the disease occurs suddenly,” he says. “That may be why these diseases usually appear in adults. The same is true with ALS, or Lou Gehrig’s disease. Gehrig was a spectacular athlete who lost the use of his muscles in the prime of life and ended up paralyzed.”
If it turns out that aggregate proteins are the toxic agents that kill cells, then one treatment would be to dissolve the aggregates or prevent them from forming, notes Kopito.
“Our study shows that a cell turns brightly fluorescent when the proteasome shuts down. That’s simple. But the study doesn’t tell us why it shuts down,” says Bence.
“We believe there are complex causes for the impairment of the proteasome,” adds Kopito. “A likely cause is a problem inside the proteasome that prevents proteins from passing through. Imagine a rope with a knot in it. The aggregate can function very much like a knot. Other studies have shown that proteins that do not unfold properly can get stuck inside the proteasome. We want to conduct another experiment to see if aggregates get stuck, too.”
In addition to Bence and Kopito, the May Science study was co-authored by Roopal M. Sampat, a graduating senior in biological sciences at Stanford.
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