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

Biochemists identify how tissue cells detect and perfect

Scientists have discovered how cells detect tissue damage and modify their repair properties accordingly. The findings, published today [6 October] in the journal Developmental Cell, could open up new opportunities for improving tissue repair in patients following illness or surgery.

The Wellcome Trust-funded study, led by biochemists at the University of Bristol, examined the signalling process in damaged tissue cells and identified the cellular mechanisms responsible for activating effective repair.

In healthy adults the majority of tissue cells lie dormant unless challenged by wounding, at which point they sense a change in the molecular environment. Plasma leaking from damaged blood vessels and causes fibroblast cells to migrate into the damaged tissue, contract the wound, and plug the gap by depositing a substance such as collagen, which provides the structural support.

Dr Mark Bass, lead author and Research Fellow in the University's School of Biochemistry, said: "Each of these processes requires the turnover of cellular adhesions, and the challenge has been to determine how cells detect tissue damage and modify their adhesive properties accordingly."

Using atomic force microscopy, the team were able to determine how a molecule sensor, syndecan-4, triggers the uptake and redeployment of adhesive molecules. This novel signalling pathway causes fibroblasts and keratinocytes to migrate in response to the changing tissue architecture and follow the matrix fibres that make up the skin. Such linear migration towards a damage signal allows the cells to arrive at the wound far more efficiently than if activated cells searched randomly about the tissue, and results in a very efficient healing response.

Dr Bass added: "We find that this signalling cascade is essential for efficient healing, this opens up considerable opportunities for improving tissue repair in patients."

More information: The Wellcome Trust-funded study, entitled 'A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis' by Dr Mark Bass is published in the journal Developmental Cell.

Provided by University of Bristol (news : web)

Biochemists identify new genetic code repair tool

Clemson University researchers recently reported finding a new class of DNA repair-makers.

Clemson biochemist Weiguo Cao studies how cells repair damaged DNA. The finding from Cao's lab in the Clemson Biosystems Research Complex in collaboration with computational chemist Brian Dominy appeared in the Sept. 9 issue of The Journal of Biological Chemistry: "A new family of deamination repair enzymes in the uracil DNA glycosylase superfamily by Hyun-Wook Lee, Brian N. Dominy and Weiguo Cao."

"DNA is a string of a long molecule composed of four building blocks: A for adenine, T for thymine, G for guanine and C for cytosine. The heredity of all organisms is determined by the pairing of A with T and G with C," said Cao, a professor in the genetics and biochemistry department.

DNA is constantly assaulted by various stresses. A common type of damage is modification of three out of the four building blocks for genetic code, A, G, C by a chemical process called deamination. The genetic consequence of deamination is that it will change the pairing of the genetic code. For example, the deamination of C (cytosine) will generate U (uracil). Instead of pairing with G as C will do, U pairs with A. In so doing, it changes the genetic program inside the cell and may cause dangerous mutations resulting in disease.

To ensure the integrity of the genetic material, cells are equipped with a "molecular toolkit" for repairing DNA damage. The toolkit is comprised of a variety of different molecules — called enzymes — that have evolved to repair different types of DNA damage. One of the DNA repair enzymes the Cao lab studies is called uracil DNA glycosylase (UDG). As it's name indicates, it is traditionally known as an enzyme that removes uracil from DNA. Because deamination of C (cytosine) is a very common type of damage found in DNA, UDG has been found in many organisms and researchers have grouped them into five families in the so-called UDG superfamily.

In their most recent work, Cao and his colleagues discovered a new class of enzymes in that superfamily that lack the ability to repair uracil. A further study showed that this class of enzymes, instead, is engaged in the repair of deamination on the different building block adenine. This caught them by surprise because all known UDG enzymes are capable of uracil repair.

To further understand how this new class of enzymes works as a tool for repair, Cao and Dominy combined computational and biochemical methods to pinpoint the critical part of the repair machine that is responsible.

"What we learned from this work is that DNA repair toolkits have an amazing ability to evolve different repair functions for different kinds of DNA damage," Cao said. "This work also demonstrates how a combination of research approaches from different disciplines makes the discovery possible."

"Collaborative efforts involving computational and experimental investigative methods can greatly enhance the efficiency of scientific discovery, as well as provide more thorough answers to very important scientific questions," Dominy said. "In my opinion, the collaborative efforts between our two groups have demonstrated the substantial value of such interactions."

Provided by Clemson University (news : web)

Biochemists reveal new twist on old fuel source

Stinging from humiliating defeat in World War I, Germany’s Nazi regime seized on technology developed by chemists Franz Fischer and Hans Tropsch that enabled the coal-rich, petroleum-poor country to produce synthetic fuels for its military machine. Research in Fischer-Tropsch or “FT” synthesis waned in the latter half of the 20th century but, like “a bubblin’ crude,” has resurfaced in recent years with growing interest in alternative fuels.

While studying bacterial enzymes, known as nitrogenases, used in nitrogen reduction, Utah State University biochemists Zhi-Yong Yang and Lance Seefeldt, along with colleague Dennis Dean of Virginia Tech, discovered a molybdenum nitrogenase capable of converting carbon monoxide into usable hydrocarbons. The reaction is similar, they say, to FT synthesis.

“This is pretty profound,” says Seefeldt, professor in USU’s Department of Chemistry and Biochemistry. “Understanding this process paves the way for developing better ways of converting carbon monoxide, a toxic waste product of combustion, into transportation fuel and precursors for plastics – without the time and energy required for conventional extraction of fossil fuels.”

The scientists’ findings appear in the article “Molybdenum Nitrogenase Catalyzes the Reduction and Coupling of CO to Form Hydrocarbons,” in the June 3, 2011 issue (and May 27 online issue) of Journal of Biological Chemistry. The paper was selected as “Paper of the Week” by the journal’s editorial board, an honor bestowed on the top one percent of more than 6,600 manuscripts reviewed annually

by the publication’s editors. In the “Paper of the Week” feature, Yang, a doctoral candidate mentored by Seefeldt, is highlighted as an up-and-coming researcher.

Molybdenum, often called “Moly,” is a brittle, silver-gray metal found in soil and used in steel alloys. It’s also found, in small amounts, in the human body, where it metabolizes certain amino acids, produces uric acid and helps to break down drugs and toxins.

“There’s tremendous interest in converting various kinds of waste into fuel and, especially, in finding cost-effective and environmentally clean ways to do it,” says Yang, who earned his first doctorate in organic chemistry at China’s Nankai University.

Unlike coal, Fischer and Tropsch’s original source for synthetic fuels, carbon monoxide produces hydrocarbons with much less pollution. The substance provides an added benefit: it allows scientists to produce longer chain, double and triple-bond hydrocarbons, which provides a richer feedstock for production of refined transportation fuels.

“Like many waste-to-energy processes, we’ve found we can produce such hydrocarbons as propane and butane from carbon monoxide,” Yang says. “But using this process, we may have the potential to produce such transportation fuels as diesel and gasoline that are readily adaptable to today’s vehicles.”

Dinitrogen, Seefeldt says, makes up about 80 percent of the air we breathe. Though essential for all life on the planet, it’s not in a form higher organisms can directly access.

“It’s kind of like being hungry and sitting at a table laden with food but not being able to eat,” he says.

Humans and animals take in nitrogen – in the form of protein – from food; plants obtain nitrogen from soil.

In recent years, Seefeldt has identified key steps involved in nitrogen fixation, the process by which nitrogen is converted to ammonia. The findings contribute to research that could enable an alternative, clean method of producing nitrogen.

Science and industry currently rely on the century-old Häber-Bosch process to produce nitrogen for fertilizer, paper, pharmaceuticals, plastics, mining and explosives. Developed by German Nobel Prize winner Fritz Häber and Carl Bosch during World War I, the process, Seefeldt says, is costly and energy-intensive.

Provided by Utah State University