Scientists discover new way protein degradation is regulated

Chamber of doom. Rockefeller scientists have identified a new way that proteins are degraded in the proteasome (green). They found that the enzyme tankyrase regulates proteasome activity by promoting the assembly of proteasome subunits into the active complex called 26S. Credit: Image by Sigi Benjamin-Hong, Strang Laboratory of Apoptosis and Cancer Biology

Proteins, unlike diamonds, aren't forever. And when they wear out, they need to be degraded in the cell back into amino acids, where they will be recycled into new proteins. Researchers at Rockefeller University and the Howard Hughes Medical Institute have identified a new way that the cell's protein recycler, the proteasome, takes care of unwanted and potentially toxic proteins, a finding that has implications for treating muscle wasting, neurodegeneration and cancer.

The consensus among scientists has been that the proteasome is constantly active, chewing up proteins that have exceeded their shelf life. A mounting body of evidence now suggests that the proteasome is dynamically regulated, ramping up its activity when the cell is challenged with especially heavy protein turnover. The researchers, postdoctoral associate Park F. Cho-Park and Hermann Steller, head of the Strang Laboratory of Apoptosis and Cancer Biology at Rockefeller, have shown that an enzyme called tankyrase regulates the proteasome's activity. In addition, Cho-Park and Steller demonstrate that a small molecule called XAV939, originally identified by scientists at Novartis who developed it as therapeutic for colon cancer, inhibits tankyrase and blocks the proteasome's activity. The research is reported in today's issue of the journal Cell.

"Our findings have tremendous implications for the clinic since it gives a new meaning to an existing class of small-molecule compound," says Steller, Strang Professor at Rockefeller and an investigator at HHMI. "In particular, our work suggests that tankyrase inhibitors may be clinically useful for treating multiple myeloma."

Tankyrase was originally identified in the late 1990s by Rockefeller's Titia de Lange and her colleagues in the Laboratory for Cell Biology and Genetics, who showed that it plays a role in elongating telomeres, structures that cap and protect the ends of chromosomes. In a series of experiments in fly and human cells, Cho-Park and Steller discovered that tankyrase uses a process called ADP-ribosylation to modify PI31, a key factor that regulates the activity and assembly of proteasome subunits into the active complex called 26S. By promoting the assembly of more 26S particles, cells under stress can boost their ability to break down and dispose of unwanted proteins.

The proteasome is currently a target for developing cancer therapeutics. The FDA has approved Velcade, a proteasome inhibitor, for the treatment of multiple myeloma and mantle cell lymphoma. However, patients on Velcade can experience peripheral neuropathy or become resistant to the drug.

Multiple myeloma cells need increased proteasome activity to survive. Preliminary data from Cho-Park and Steller show that XAV939 can block the growth of multiple myeloma cells by inhibiting the assembly of additional proteasomes without affecting the basal level of proteasomes in the cell. This selective targeting may mean fewer side effects for patients. 

"Drugs, such as XAV939, that inhibit the proteasome through other mechanisms than Velcade may have significant clinical value," says Steller.

The findings by Cho-Park and Steller also link, for the first time, metabolism and regulation of the proteasome. Sometimes the proteasome digests too much protein, which can lead to loss of muscle, says Steller.

"This discovery reveals fundamental insights into protein degradation, a process important for normal cell biology, and a key factor in disorders such as muscle wasting and neurodegeneration," said Stefan Maas of the National Institutes of Health's National Institute of General Medical Sciences, which partly supported the study. "Intriguingly, the findings also enlighten ongoing research on cancer therapies, exemplifying the impact of basic research on drug development."

Source: Rockefeller University

ADHD: Brains not recognizing angry expressions

These two faces were presented to children. Credit: © National Institutes of Natural Sciences

Inattention, hyperactivity, and impulsive behavior in children with ADHD can result in social problems and they tend to be excluded from peer activities. They have been found to have impaired recognition of emotional expression from other faces.

The research group of Professor Ryusuke Kakigi of the National Institute for Physiological Sciences, National Institutes of Natural Sciences, in collaboration with Professor Masami K. 

Yamaguchi and Assistant Professor Hiroko Ichikawa of Chuo University first identified the characteristics of facial expression recognition of children with ADHD by measuring hemodynamic response in the brain and showed the possibility that the neural basis for the recognition of facial expression is different from that of typically developing children.

The findings are discussed in Neuropsychologia.

The research group showed images of a happy expression or an angry expression to 13 children with ADHD and 13 typically developing children and identified the location of the brain activated at that time. They used non-invasive near-infrared spectroscopy to measure brain activity. Near-infrared light, which is likely to go through the body, was projected through the skull and the absorbed or scattered light was measured. The strength of the light depends on the concentration in "oxyhemoglobin" which gives the oxygen to the nerve cells working actively. The result was that typically developing children showed significant hemodynamic response to both the happy expression and angry expression in the right hemisphere of the brain.

On the other hand, children with ADHD showed significant hemodynamic response only to the happy expression but brain activity specific for the angry expression was not observed. 

This difference in the neural basis for the recognition of facial expression might be responsible for impairment in social recognition and the establishment of peer-relationships.

Source: National Institutes of Natural Sciences

ALS progression linked to increased protein instability

The new study provides evidence that proteins linked to more severe forms of ALS are less stable structurally and more prone to form clusters or aggregates. Mutants of the superoxide dismutase (SOD) protein formed long, rod-shaped aggregates (shown here as red lattice), compared to the compact folded structure of wild-type SOD (purple ribbons). Credit: Image courtesy of the Getzoff and Tainer labs, The Scripps Research Institute.

A new study by scientists from The Scripps Research Institute (TSRI), Lawrence Berkeley National Laboratory (Berkeley Lab) and other institutions suggests a cause of amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease.

"Our work supports a common theme whereby loss of protein stability leads to disease," said John A. Tainer, professor of structural biology at TSRI and senior scientist at Berkeley Lab, who shared senior authorship of the new research with TSRI Professor Elizabeth Getzoff.

Getzoff, Tainer and their colleagues, who focused on the effects of mutations to a gene coding for a protein called superoxide dismutase (SOD), report their findings this week in the online Early Edition of the Proceedings of the National Academy of Sciences. The study provides evidence that those proteins linked to more severe forms of the disease are less stable structurally and more prone to form clusters or aggregates.

"The suggestion here is that strategies for stabilizing SOD proteins could be useful in treating or preventing SOD-linked ALS," said Getzoff.

Striking in the Prime of Life

ALS is notorious for its ability to strike down people in the prime of life. It first leapt into public consciousness when it afflicted baseball star Lou Gehrig, who succumbed to the disease in 1941 at the age of only 38. Recently, the ALS Association's Ice Bucket Challenge has enhanced public awareness of the disease.

ALS kills by destroying muscle-controlling neurons, ultimately including those that control breathing. At any one time, about 10,000 Americans are living with the disease, according to new data from the Centers for Disease Control and Prevention, but it is almost always lethal within several years of the onset of symptoms.

SOD1 mutations, the most studied factors in ALS, are found in about a quarter of hereditary ALS cases and seven percent of ordinary "sporadic" ALS cases. SOD-linked ALS has nearly 200 variants, each associated with a distinct SOD1 mutation. Scientists still don't agree, though, on just how the dozens of different SOD1 mutations all lead to the same disease.

One feature that SOD1-linked forms of ALS do have in common is the appearance of SOD clusters or aggregates in affected motor neurons and their support cells. Aggregates of SOD with other proteins are also found in affected cells, even in ALS cases that are not linked to SOD1 mutations.

In 2003, based on their and others' studies of mutant SOD proteins, Tainer, Getzoff and their colleagues proposed the "framework destabilization" hypothesis. In this view, ALS-linked mutant SOD1 genes all code for structurally unstable forms of the SOD protein. 
Inevitably some of these unstable SOD proteins lose their normal folding enough to expose sticky elements that are normally kept hidden, and they begin to aggregate with one another, faster than neuronal cleanup systems can keep up -- and that accumulating SOD aggregation somehow triggers disease.

Faster Clumping, Worse Disease

In the new study, the Tainer and Getzoff laboratories and their collaborators used advanced biophysical methods to probe how different SOD1 gene mutations in a particular genetic ALS "hotspot" affect SOD protein stability.

To start, they examined how the aggregation dynamics of the best-studied mutant form of SOD, known as SOD G93A, differed from that of non-mutant, "wild-type" SOD. To do this, they developed a method for gradually inducing SOD aggregation, which was measured with an innovative structural imaging system called SAXS (small-angle X-ray scattering) at Berkeley Lab's SIBYLS beamline.

"We could detect differences between the two proteins even before we accelerated the aggregation process," said David S. Shin, a research scientist in Tainer's laboratories at Berkeley Lab and TSRI who continues structural work on SOD at Berkeley.

The G93A SOD aggregated more quickly than wild-type SOD, but more slowly than an SOD mutant called A4V that is associated with a more rapidly progressing form of ALS.

Subsequent experiments with G93A and five other G93 mutants (in which the amino acid glycine at position 93 on the protein is replaced with a different amino acid) revealed that the mutants formed long, rod-shaped aggregates, compared to the compact folded structure of wild-type SOD. The mutant SOD proteins that more quickly formed longer aggregates were again those that corresponded to more rapidly progressing forms of ALS.

What could explain these SOD mutants' diminished stability? Further tests focused on the role of a copper ion that is normally incorporated within the SOD structure and helps stabilize the protein. Using two other techniques, electron-spin resonance (ESR) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS), the researchers found that the G93-mutant SODs seemed normal in their ability to take up copper ions, but had a reduced ability to retain copper under mildly stressing conditions -- and this ability was lower for the SOD mutants associated with more severe ALS.

"There were indications that the mutant SODs are more flexible than wild-type SOD, and we think that explains their relative inability to retain the copper ions," said Ashley J. Pratt, the first author of the study, who was a student in the Getzoff laboratory and postdoctoral fellow with Tainer at Berkeley Lab.

Toward New Therapies

In short, the G93-mutant SODs appear to have looser, floppier structures that are more likely to drop their copper ions -- and thus are more likely to misfold and stick together in aggregates.

Along with other researchers in the field, Getzoff and Tainer suspect that deviant interactions of mutant SOD trigger inflammation and disrupt ordinary protein trafficking and disposal systems, stressing and ultimately killing affected neurons.

"Because mutant SODs get bent out of shape more easily," said Getzoff, "they don't hold and release their protein partners properly. By defining these defective partnerships, we can provide new targets for the development of drugs to treat ALS."

The researchers also plan to confirm the relationship between structural stability and ALS severity in other SOD mutants.

"If our hypothesis is correct," said Shin, "future therapies to treat SOD-linked ALS need not be tailored to each individual mutation -- they should be applicable to all of them."

Source: The Scripps Research Institute

Multiple allergic reactions traced to single protein

This is a mast cell. Credit: Priyanka Pundir/University of Alberta

Johns Hopkins and University of Alberta researchers have identified a single protein as the root of painful and dangerous allergic reactions to a range of medications and other substances. If a new drug can be found that targets the problematic protein, they say, it could help smooth treatment for patients with conditions ranging from prostate cancer to diabetes to HIV. Their results appear in the journal Nature on Dec. 17.

Previous studies traced reactions such as pain, itching and rashes at the injection sites of many drugs to part of the immune system known as mast cells. When specialized receptors on the outside of mast cells detect warning signals known as antibodies, they spring into action, releasing histamine and other substances that spark inflammation and draw other immune cells into the area. Those antibodies are produced by other immune cells in response to bacteria, viruses or other perceived threats. However, "although many of these injection site reactions look like an allergic response, the strange thing about them is that no antibodies are produced," says Xinzhong Dong, Ph.D., an associate professor of neuroscience in the Institute for Basic Biomedical Sciences at the Johns Hopkins University School of Medicine.

To zero in on the cause of the reactions, Benjamin McNeil, Ph.D., a postdoctoral fellow in Dong's laboratory, first set out to find which mast cell receptor -- or receptors -- responded to the drugs in mice. Previous studies had identified a human receptor likely to be at fault in the allergic reactions; McNeil found a receptor in mice that, like the human receptor, is found only in mast cells. He then tested that receptor by putting it into lab-grown cells and found that they did react to medications that provoke mast cell response. He found similar results for the human receptor that previous studies had indicated was a likely culprit.

"It's fortunate that all of the drugs turn out to trigger a single receptor -- it makes that receptor an attractive drug target," McNeil says.

To find out whether eliminating the receptor really would eliminate the allergic reactions, the research team also disabled the gene for the suspect receptor in mice. These "knockout" mice did not have any of the drug allergy symptoms that their genetically normal counterparts displayed.

The researchers are now working to find compounds that could safely block the culprit receptor in humans, known as MRGPRX2. Such a drug would not prevent true allergic reactions, which produce antibodies, but only the pseudoallergic reactions triggered by MRGPRX2. Still, it could improve the lives of many patients, says McNeil, by lessening the drug side effects they currently endure. Medications that trigger MRGPRX2 include cancer drugs cetrorelix, leuprolide and octreotide; HIV drug sermorelin; fluoroquinolone antibiotics; and neuromuscular blocking drugs used to paralyze muscles during surgeries.

Dong's research group is also looking into the possibility that MRGPRX2 could be behind immune conditions such as rosacea and psoriasis that don't stem from medication use.

Source: Johns Hopkins Medicine