Biochemists develop promising new treatment direction for rare metabolic diseases

People born with Fabry disease have a faulty copy of a single gene that codes for the alpha-galactosidase (?-GAL) enzyme, one of the cell's "recycling" machines. When it performs normally, ?-GAL breaks down an oily lipid known as GB3 in the cell's recycling center, or lysosome. But when it underperforms or fails, Fabry symptoms result. Patients may survive to adulthood, but the disorder leads to toxic lipid build-up in blood vessels and organs that compromise kidney function or lead to heart disease, for example.

The faulty gene causes its damage by producing a misfolded protein, yielding an unstable, poorly functioning ?-GAL enzyme. Like origami papers, these proteins are unfolded to start and only become active when folded into precise shapes. At present, enzyme replacement therapy (ERT) is the only FDA-approved treatment for such lysosomal storage disorders as Fabry, Pompe and Gaucher diseases, but ERT requires a complicated and expensive process to purify and replace the damaged ?-GAL enzyme, and it must be administered by a physician.

Instead of replacing the damaged enzyme, an alternative route called pharmacological chaperone (PC) therapy is currently in Phase III clinical trials for Fabry disease. It relies on using smaller, "chaperone" molecules to keep proteins on the right track toward proper folding, but their biochemical mechanism is not well understood, says Garman.

Now, he and colleagues report results of a thorough exploration at the atomic level of the biochemical and biophysical basis of two small molecules for potentially stabilizing the ?-GAL enzyme. He says their use in PC therapy could one day be far less expensive than the current standard, ERT, and can be taken orally.

This work, which improves knowledge of a whole class of molecular chaperones, represents the centerpiece of UMass Amherst student Abigail Guce's doctoral thesis and was supported by the National Institutes of Health. Other members of the team are graduate students Nat Clark and Jerome Rogich.

"The interactions we looked at are exactly the things occurring in the clinical trial right now," Garman says. Further, "the same concept is now being applied to other protein-folding diseases such as Parkinson's and Alzheimer's disease. Many medical researchers are trying to keep proteins from misfolding by using small chaperone molecules. Our studies have definitely advanced the understanding of how to do that."

In their current paper, Garman and colleagues compare the ability of two small chaperone molecules, galactose and 1-deoxygalactononjirimycin (DGJ) to stabilize the ?-GAL protein, to help it resist unfolding in different conditions such as high temperature and different pH levels.

They found that each chaperone has very different affinities: DGJ binds tightly and galactose binds loosely to the ?-GAL, yet they differ in only two atomic positions. "Tight is better, because you can use less drug for treatment," Garman says. "We now can explain DGJ's high potency, its tight binding, down to individual atoms."

In earlier studies as in the current work, the UMass Amherst team used their special expertise in X-ray crystallography to create three-dimensional images of all atoms in the protein to understand how it carries out its metabolic mission. They also found a new binding site for small molecules on human ?-GAL that had never been observed before.

Crystallography on the two chaperones bound to the ?-GAL enzyme showed that a single interaction between the enzyme and DGJ was responsible for DGJ's high affinity for the enzyme. Other experiments also showed the ability of the 11- and 12-atom chaperones to protect the large, 6,600-atom ?-GAL from unfolding and degradation.

For the first time, by making a single change in one amino acid in protein, they forced the DGJ to bind weakly, indicating that one atomic interaction is responsible for DGJ's high affinity.

"It was surprising to find these two small molecules that look very much the same have very different affinities for this enzyme," says Garman, "and we now understand why. The iminosugar DGJ has high potency due to a single ionic interaction with ?-GAL. Overall, our studies show that this small molecule keeps the enzyme from unfolding, or when it unfolds, the process happens more slowly, all of which you need in treating disease."

Provided by University of Massachusetts at Amherst

Scientists discover new direction in Alzheimer's research

In what they are calling a new direction in the study of Alzheimer's disease, UC Santa Barbara scientists have made an important finding about what happens to brain cells that are destroyed in Alzheimer's disease and related dementias. The results are published in the online version of The Journal of Biological Chemistry.

Stuart Feinstein, professor of Molecular, Cellular and Developmental Biology, senior author, and co-director of UCSB's Neuroscience Research Institute, explained: "With dementia, the brain cells, or neurons, that you need for cognitive skills are no longer working properly. Then, they're not even there anymore because they die. That's what leads to dementia; you're losing neuronal capacity."

Feinstein has studied the protein called "tau" for about 30 years, using test tube biochemistry and a variety of cultured cells as models. Under normal conditions, tau is found in the long axons of neurons that serve to connect neurons with their targets, often far from the cell body itself. Among tau's major functions is to stabilize microtubules, which are an integral part of the cellular cytoskeleton that is essential for many aspects of neuronal cell structure and function.

It has been known for many years that a small peptide named amyloid beta can cause neuronal cell death and Alzheimer's disease, although the mechanism for how it works has been poorly understood. Recently, genetic evidence has demonstrated that the ability of amyloid beta to kill neurons requires tau; however, what it does to tau has been enigmatic. "We know amyloid beta is a bad guy," said Feinstein. "Amyloid beta causes disease; amyloid beta causes Alzheimer's. The question is how does it do it?"

He explained that most Alzheimer's researchers would argue that amyloid beta causes tau to become abnormally and excessively phosphorylated. This means that the tau proteins get inappropriately chemically modified with phosphate groups. "Many of our proteins get phosphorylated," said Feinstein. "It can be done properly or improperly."

Feinstein added that he and his students wanted to determine the precise details of the presumed abnormal phosphorylation of tau in order to gain a better understanding of what goes wrong. "That would provide clues for drug companies; they would have a more precise target to work on," said Feinstein. "The more precisely they understand the biochemistry of the target, the better attack a pharmaceutical company can make on a problem."

Feinstein said that the team's initial hypothesis suggesting that amyloid beta leads to extensive abnormal tau phosphorylation turned out not to be true. "We all like to get a curve ball tossed our way once in a while, right?" said Feinstein. "You like to see something different and unexpected."

The research team found that when they added amyloid beta to neuronal cells, the tau in those cells did not get massively phosphorylated, as predicted. Rather, the surprising observation was the complete fragmentation of tau within one to two hours of exposure of the cells to amyloid beta. Within 24 hours, the cells were dead.

Feinstein explained that tau has many jobs, but its best-understood job is to regulate the cellular cytoskeleton. Cells have a skeleton much like humans have a skeleton. The major difference is that human skeletons don't change shape very abruptly, whereas a cell's skeleton is constantly growing, shortening, and moving. It does this in order to help the cell perform many of its essential functions. The cytoskeleton is especially important to neurons because of their great length.

Feinstein argues that neurons die in Alzheimer's disease because their cytoskeleton is not working properly. "If you destroy tau, which is an important regulator of the microtubules, one could easily see how that could also cause cell death," said Feinstein. "We know from cancer drugs that if you treat cells with drugs that disrupt the cytoskeleton, the cells die," he said. "In my mind, the same thing could be happening here."

Provided by University of California - Santa Barbara (news : web)

'Lost' samples from famous origin of life researcher could send search for first life in new direction

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Preserved samples from a 1958 experiment done by "primordial soup" pioneer Stanley Miller contain amino acids created by the experiment. The samples had not undergone analysis until recently when Miller's former student Jeffrey Bada and colleagues discovered a wide range of amino acids. The find could be an important step toward understanding how life on Earth could have originated. The vials have been relabeled but the boxes are marked with Miller's original notes. Credit: Scripps Institution of Oceanography, UC San Diego

(PhysOrg.com) -- Stanley Miller gained fame with his 1953 experiment showing the synthesis of organic compounds thought to be important in setting the origin of life in motion. Five years later, he produced samples from a similar experiment, shelved them and, as far as friends and colleagues know, never returned to them in his lifetime.

More 50 years later, Jeffrey Bada, Miller's former student and a current Scripps Institution of Oceanography, UC San Diego professor of marine chemistry, discovered the samples in Miller's laboratory material and made a discovery that represents a potential breakthrough in the search for the processes that created Earth's first life forms.

Former Scripps undergraduate student Eric Parker, Bada and colleagues report on their reanalysis of the samples in the March 21 issue of Proceedings of the National Academy of Sciences. Miller's 1958 experiment in which the gas hydrogen sulfide was added to a mix of gases believed to be present in the atmosphere of early Earth resulted in the synthesis of sulfur amino acids as well as other amino acids. The analysis by Bada's lab using techniques not available to Miller suggests that a diversity of organic compounds existed on early planet Earth to an extent scientists had not previously realized.

 

Scripps Oceanography professor of Marine Chemistry Jeffrey Bada holds a preserved sample from a 1958 experiment done by "primordial soup" pioneer Stanley Miller. The residue in the sample contains amino acids created by the experiment. The samples had not undergone analysis until recently when Bada and colleagues discovered a wide range of amino acids using modern detection methods. Credit: Scripps Institution of Oceanography, UC San Diego

The new findings support the case that volcanoes — a major source of atmospheric hydrogen sulfide today — accompanied by lightning converted simple gases into a wide array of amino acids, which are were in turn available for assembly into early proteins.

Bada also found that the amino acids produced in Miller's experiment with hydrogen sulfide are similar to those found in meteorites. This supports a widely-held hypothesis that processes such as the ones in the laboratory experiments provide a model of how organic material needed for the origin of life are likely widespread in the universe and thus may provide the extraterrestrial seeds of life elsewhere.

Successful creation of the sulfur-rich amino acids would take place in the labs of several researchers, including Miller himself, but not until the 1970s.

"Unbeknownst to him, he'd already done it in 1958," said Bada.

Miller's initial experiments in the 1950s with colleague Harold Urey used a mixture of gases such as methane, ammonia, water vapor and hydrogen and electrically charged them as lightning would. The experiment, which took place in a closed chamber meant to simulate conditions on early Earth, generated several simple amino acids and other organic compounds in what became known as "primordial soup."

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This is a photo of Stanley Miller in his UC San Diego lab in 1970. Credit: Scripps Institution of Oceanography Archives

With the gases and electrical energy they produce, many geoscientists believe the volcanoes on a young planet covered much more extensively by water than today's served as oases of raw materials that allowed prebiotic matter to accumulate in sufficient quantities to assemble into more complex material and eventually into primitive life itself. Bada had already begun reanalyzing Miller's preserved samples and drawing conclusions about the role of volcanoes in sparking early life when he came across the previously unknown samples. In a 2008 analysis of samples left from Miller's more famous experiment, Bada's team had been able to detect many more amino acids than his former mentor had thanks to modern techniques unavailable to Miller.

Miller, who became a chemistry professor at UCSD in 1960, conducted the experiments while a faculty member at Columbia University. He had collected and catalogued samples from the hydrogen sulfide mix but never analyzed them. He only casually mentioned their existence late in his life and the importance of the samples was only realized shortly before his death in 2007, Bada said. It turned out, however, that his 1958 mix more closely resembled what geoscientists now consider early Earth conditions than did the gases in his more famous previous experiment.

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The original box containing archived spark discharge samples prepared by Stanley Miller in 1958. For unknown reasons, Miller never analyzed these even though this is his first experiment using hydrogen sulfide. The label shows Miller?s original writing: p 114 refers to his notebook. Credit: Jeffrey Bada and Robert Benson/Scripps Institution of Oceanography, University of California at San Diego

"This really not only enhances our 2008 study but goes further to show the diversity of compounds that can be produced with a certain gas mixture," Bada said.

The Bada lab is gearing up to repeat Miller's classic experiments later this year. With modern equipment including a miniaturized microwave spark apparatus, experiments that took the elder researcher weeks to carry out could be completed in a day, Bada said.

Provided by University of California - San Diego (news : web)