What happens when a super long slinky is dropped?
Thursday, October 13, 2011
If oxygen becomes the undoing of proteins
Scientists from the Faculty of Biology and Biotechnology at the RUB have published a report in the Journal of Biological Chemistry explaining why enzymes used for the production of hydrogen are so sensitive to oxygen. In collaboration with researchers from Berlin, they used spectroscopic methods to investigate the time course of the processes that lead to the inactivation of the enzyme’s iron center. “Such enzymes, the so-called hydrogenases, could be extremely significant for the production of hydrogen with the help of biological or chemical catalysts”, explains Camilla Lambertz from the RUB study group for photobiotechnology. “Their extreme sensitivity to oxygen is however a major problem. In future, our results could help to develop enzymes that are more robust.”
Oxygen as a friend and as an enemy
Oxygen is crucial for the survival of most animals and plants. It is however toxic for many living creatures if the concentration thereof is too high, and some organisms can even only exist entirely without oxygen. Sensitivity to oxygen is also present at the protein level. A large number of enzymes, for example, hydrogenases are known to be irreversibly destroyed by oxygen. Hydrogenases are biological catalysts that convert protons and electrons into technically usable hydrogen. The RUB team of Prof. Thomas Happe is doing research on so-called [FeFe]-hydrogenases which are capable of producing particularly large amounts of hydrogen. The generation of hydrogen takes place at the H-cluster, consisting of a di-iron and four-iron subcluster which, together with other ligands, form the reactive center.
Oxygen attacks the iron centers
The researchers, working in collaboration with Dr. Michael Haumann’s team in Berlin, discovered that oxygen binds to the di-iron center of the hydrogenase, which initiates the inactivation of another part of the enzyme consisting of four further iron atoms. In this project, sponsored by the BMBF, it was possible to show the diverse phases of the inactivation process for the first time using the so-called X-ray absorption spectroscopy. The researchers used the synchroton radiation source Swiss Light Source in Switzerland for this specific type of measurement. It generates particularly strong rays, thus enabling the characterization of metal centers in proteins. Amongst other things, the scientists thus determined the chemical nature of the iron centers and the distance from the surrounding atoms using atomic resolution.
Inactivation in three phases
The team of researchers from Bochum and Berlin used a new experimental procedure. They initially brought the hydrogenase sample into contact with oxygen for a few seconds to minutes and finally for a couple of hours and then suppressed all proceeding reactions by deep-freezing it in liquid nitrogen. The subsequently gained spectroscopic data was used for the development of a model for a three-phase inactivation process. According to this model, an oxygen molecule initially binds to the di-iron center of the hydrogenase, which leads to the development of an aggressive oxygen species. In the subsequent phase, this attacks and modifies the four-iron center. During the final phase, further oxygen molecules bind and the entire complex disintegrates. “The entire process thus consists of a number of consecutive reactions that are distinctly separated in time”, says Lambertz. “The velocity of the entire process is possibly dependent on the phase during which the aggressive oxygen species moves from the di-iron to the four-iron center. We are currently elaborating further experiments to investigate this.”
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Title:
C. Lambertz, N. Leidel, K.G.V. Havelius, J. Noth, P. Chernev, M. Winkler, T. Happe, M. Haumann (2011) O2-reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase, Journal of Biological Chemistry, doi: 10.1074/jbc.M111.283648
Researchers develop new way to screen for brain cancer stem cell killers
Researchers with UCLA’s Jonsson Comprehensive Cancer Center have developed and used a high-throughput molecular screening approach that identifies and characterizes chemical compounds that can target the stem cells that are responsible for creating deadly brain tumors.
Glioblastoma is one of the deadliest malignancies, typically killing patients within 12 to 18 months. These brain cancers consist of two kinds of cells, a larger, heterogeneous population of tumor cells and a smaller sub-population of stem cells, which are treatment-resistant.
The screening system was specifically designed to find drugs that can target that sub-population and prevent it from re-seeding the brain cancer, said study senior author Dr. Harley Kornblum, a Jonsson Cancer Center scientist and a professor of psychiatry and biobehavioral sciences.
“We’re pleased that we can present a different way to approach the discovery of potential new cancer drugs,” said Kornblum, who also is a researcher with the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. “And by finding these drugs, we may be able to reveal things about the biology of these cancer stem cells.”
The study appears in the Oct. 10 issue of Molecular Cancer Therapeutics, a peer-reviewed journal of the American Association of Cancer Research.
After testing more than 31,000 compounds from seven chemical libraries in an initial screen, the team came up with 694 that showed some activity against the brain cancer stem cells. After further narrowing the field down to 168 compounds, they decided to focus on four in future studies because they most successfully inhibited the brain cancer stem cells, Kornblum said.
What Kornblum and his team did in their approach was sort of a reverse of the usual screening processes. Typically, researchers doing high-throughput screening are seeking a drug to hit a specific target they know is on a cancer cell, perhaps a protein that is causing it to grow or a gene that keeps it from dying. In this case, Kornblum said, the team was basically shooting in the dark because the biology of these brain cancer stem cells is largely unknown.
“When brain cancer stem cells were first discovered, we all realized rapidly that we would need to find drugs that attack these cells specifically, because they’re resistant to our conventional therapies,” Kornblum said. “We needed a way to kill these stem cells.”
UCLA’s high-throughput screening technology is capable of screening as many as 100,000 compounds in a single day. Researchers generally develop cancer cells lines and then create an assay, a procedure in molecular biology to test or measure the activity of a drug or biochemical compound in an organic sample, in this case the cancer cells.
The cells are loaded into plates with 384 wells each and the drugs are added. The plates are about the size of the palm of an adult hand. The computerized, robotic screening system executes the process from start to finish, adding the compounds sitting in the tiny wells in the plates to the cancer cells, located in corresponding assay plates.
In this study, Kornblum and his team had a few clues to help them in narrowing down potential candidates that kill brain cancer stem cells. One method they used was based on a prior discovery by Jonsson Cancer Center researchers. The researchers had identified genes that correlate with how aggressive a brain tumor is, so Kornblum decided to try to find potential drug candidates that might reduce the expression of these genes. Another approach was to figure out which of the molecules killed brain cancer stem cells with a greater potency than they attacked other cells within glioblastoma.
To grow his cell lines, Kornblum used human tissue taken from UCLA patients diagnosed with glioblastoma. He knew that a certain method of culturing brain cancer cells resulted in a large number of brain cancer stem cells in the population. These cells were then screened with a molecular library of 31,624 compounds available through the cancer center’s Molecular Screening Shared Resource. These compounds encompass a wide range of structures and therefore have the possibility of influencing virtually all cellular functions.
“We decided on this type of approach because, although we have learned a great deal about brain cancer stem cells in the past several years, we still have not discovered enough of their biology to be sure that any single target will be the right one to hit,” Kornblum said.
Going forward, Kornblum and his team will further study the four identified “lead” compounds to see if they help reveal the biology of the brain cancer stem cells and potentially result in a new and more effective therapy for these deadly brain cancers.
“One of our goals was to determine whether some compounds selectively act on glioblastoma stem cells compared to the less tumorigenic cells from the same tumor,” the study states. “This selectivity may allow for the delineation of pathways and processes that are highly important to these cells. By making sure that a drug candidate has the potential to attack these stem cells, one might ensure the highest chance of therapeutic success.”
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