Staying healthy: Experiment finds key to natural detoxifier’s reactivity

 

A chalk drawing showing the key bonds in the cytochrome intermediate compound. The shorter iron-sulfur bond enables the more reactive double-bonded oxygen atom to extend farther out on the other side and participate in an activation reaction. Credit: Michael T. Green, Elizabeth L. Onderko/ Penn State University Researchers working at the Department of Energy's SLAC National Accelerator Laboratory have discovered that a mere 9-trillionths-of-a-meter reduction in the length of a chemical bond dramatically boosts the reactivity of a family of molecules that helps keep humans and many other organisms healthy. This result, reported recently in Nature Chemistry, is expected to help scientists design and manufacture new and more effective medicines. The molecules, called cytochrome P450s, are known as "nature's detoxifiers," said Courtney Krest Roach, a beamline scientist at SLAC's Stanford Synchrotron Radiation Lightsource (SSRL) -- a DOE Office of Science User Facility -- and first author of the paper. Since cytochrome P450 was discovered more than 50 years ago, more than 11,000 variants have been identified in every kingdom of life, including animals, plants, fungi and bacteria. Some 57 different P450s are found in humans, typically in the liver, where they help break down poisons and unwanted molecules. They are also essential in the body's manufacture of certain hormones, and chemists hope to be able to use them in the future to catalyze the creation of complex molecules for use in medicine and industry. "Our bodies use P450s to perform one step in the metabolism of some 75 percent of pharmaceuticals," Roach said. "And the P450s' extreme selectivity can enable much more direct and efficient synthesis of a number of high-value hydrocarbons and organic molecules." Molecular Structure is Key Each cytochrome P450 is optimized to catalyze an initial "activation" step in a specific chemical reaction. They all share the same basic atomic structure: A central iron atom is surrounded by four nitrogen atoms, with a sulfur-based structure sticking out from one side of the ring. Activation creates an intermediate compound that has an oxygen atom double-bonded to the iron atom; it sticks out from the other side of the ring. Because the P450s' intermediate compounds were short-lived, chemists who were trying to learn how they worked tried studying a slightly simpler molecule, called CPO, which had a more stable intermediate. But they found it was much less reactive than the P450s ­- in some cases only one-fortieth as reactive. Researchers needed to know why, because a more reactive P450 can break stronger bonds, metabolize new medicines or make different final products. The new experiments at SSRL were led by Penn State University Professor Michael Green, who was Roach's PhD advisor. The research team used the absorption of X-rays by the intermediate forms of CPO and P450s to make ultraprecise measurements of their atomic structures. The results showed that the big difference in reactivity was caused by a miniscule, 9-trillionths-of-a-meter difference in the lengths of their iron-sulfur bonds. Small Changes, Big Impact Slight changes in hydrogen bonding near the central iron atom shortened the length of the P450s' iron-sulfur bond by 4 percent. This changed the distribution of nearby electrons in a way that weakened and lengthened the iron-oxygen double bond on the opposite side of the ring. This, in turn, made it easier for the oxygen atom to carry out its part in the activation process, thus increasing P450s' reactivity. "SSRL is the only place that we could do these experiments with the necessary quality," Roach said. In their "News & Views" commentary on this research published in the same issue of Nature Chemistry, University of Illinois professors Ilia Denisove and Stephen Silgar said this result is a significant breakthrough that will provide the basis for researchers to design and make new catalysts and adapt P450-based systems for use in biotechnology. Story Source: The above post is reprinted from materials provided by SLAC National Accelerator Laboratory. Note: Materials may be edited for content and length. Journal Reference: Courtney M. Krest, Alexey Silakov, Jonathan Rittle, Timothy H. Yosca, Elizabeth L. Onderko, Julio C. Calixto, Michael T. Green. Significantly shorter Fe–S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase. Nature Chemistry, 2015; 7 (9): 696 DOI: 10.1038/nchem.2306   http://www.sciencedaily.com/releases/2015/10/151007145246.htm

Scientists create synthetic membranes that grow like living cells

 

Tue, 06/30/2015 - 11:34am UC San Diego Growing cell membranes are seen in this time lapse sequence (numbers correspond to minutes of duration). Courtesy of Michael Hardy, UC San DiegoChemists and biologists at UC San Diego have succeeded in designing and synthesizing an artificial cell membrane capable of sustaining continual growth, just like a living cell. Their achievement, detailed in a paper published in this week’s issue of the Proceedings of the National Academy of Sciences, will allow scientists to more accurately replicate the behavior of living cell membranes, which until now have been modeled only by synthetic cell membranes without the ability to add new phospholipids. “The membranes we created, though completely synthetic, mimic several features of more complex living organisms, such as the ability to adapt their composition in response to environmental cues,” said Neal Devaraj, an assistant professor of chemistry and biochemistry at UC San Diego who headed the research team, which included scientists from the campus’ BioCircuits Institute. “Many other scientists have exploited the ability of lipids to self-assemble into bilayer vesicles with properties reminiscent of cellular membranes, but until now no one has been able to mimic nature’s ability to support persistent phospholipid membrane formation,” he explained. “We developed an artificial cell membrane that continually synthesizes all of the components needed to form additional catalytic membranes.” The scientists said in their paper that to develop the growing membrane they substituted a “complex network of biochemical pathways used in nature with a single autocatalyst that simultaneously drives membrane growth.” In this way, they added, “our system continually transforms simpler, higher-energy building blocks into new artificial membranes.” “Our results demonstrate that complex lipid membranes capable of indefinite self-synthesis can emerge when supplied with simpler chemical building blocks,” said Devaraj. “Synthetic cell membranes that can grow like real membranes will be an important new tool for synthetic biology and origin of life studies.” Other members of the UC San Diego team were Michael Hardy, Jun Yang and Christian Cole of the Department of Chemistry and Biochemistry and Jangir Selimkhanov and Lev Tsimring of the BioCircuits Institute. Support for the research project was provided by UC San Diego, US Army Research Laboratory, US Army Research Office and the National Science Foundation. Time-lapse video: https://www.youtube.com/watch?v=C_FqejrYjbE SOURCE: UC San Diego

New tech could find tiny RNA cancer beacons in blood

 

Tue, 06/23/2015 - 11:40am Nicole Casal Moore, University of Michigan   University of Michigan researchers have developed a technique that allows them to efficiently identify snippets of genetic information called microRNAs in blood. The advance could one day lead to a way to scan for multiple types of cancer at once with a simple blood test. In this illustration, the red, blue and black strands represent different microRNAs that, in the new technique, attach and detach to DNA, which is gray in the image. The DNA glows fluorescent when an RNA attaches and the particular pattern of blinking tells the researchers which microRNA has attached. Image: MolGraphicsCancerous tumors cast off tiny telltale genetic molecules known as microRNAs and Univ. of Michigan researchers have come up with an efficient way to detect them in blood. The researchers say their approach could open the door to a single, inexpensive blood test to simultaneously screen for multiple types of cancer—eventually perhaps more than 100 different kinds. "This could lead to technology that enables earlier detection in individuals at risk for cancer, earlier detection of recurrences in cancer survivors, and also better and earlier assessment of how well cancer therapies are working in patients," said Dr. Muneesh Tewari, the Ray and Ruth Anderson-Laurence M. Sprague Memorial research professor of internal medicine at the U-M Medical School and an associate professor of biomedical engineering in the College of Engineering. It could be years, if not a decade, before this could be available for routine clinical use. But the researchers have high hopes for their ultrasensitive technique that can pick out a single one of these nanoscale snippets in a speck of fluid. "What we have done is develop a new paradigm, a new principle for detecting any sort of RNA in blood," said Nils Walter, professor of chemistry and biophysics in the U-M College of Literature, Science, and the Arts. Walter and Tewari are the senior authors of a study on the work published in Nature Biotechnology. RNA stands for ribonucleic acid, a class of molecule whose members play important roles in building living things from their DNA blueprints. For decades, scientists thought RNA was mainly a messenger: It ferries genetic information from DNA to the sites where cells make proteins—the workhorse molecules that essentially carry out the directions encoded in our genes. But when scientists finished sequencing the human genome around 2003, they learned that 90 percent of it contains directions for making RNA. And most of that RNA is not the messenger kind that helps make proteins. "The field of biochemistry is about 100 years old," Walter said. "And for the longest time, we were focusing on proteins. It is almost as if we were studying the wrong thing. "RNA is profoundly important for understanding and manipulating mammalian and human life, yet it is arguably the least studied genetic material in the mammalian cell. We're just at the beginning of big discoveries of its functions." MicroRNA molecules, for example, are short strands that can bind to the messenger RNA, intercepting the dispatch and preventing bits of genetic code from being put into action. More than 1,000 varieties exist in our bodies. They directly or indirectly control virtually all major life processes, the researchers say. Having too little or too much of a particular microRNA can fuel tumor growth. Cancerous cells are descendent from haywire healthy ones, so they have microRNA in them too. The tiny strands of genetic material have been detected in blood before (though not very efficiently) and scientists have several hypotheses about how they get there. They may be released when a cancerous cell dies and breaks down. And cells, including cancerous ones, may communicate with one another through microRNAs they send into the bloodstream to act as hormones. Blood-borne microRNAs from both mechanisms would be the cancer beacons the new technique could efficiently detect in patients, the researchers say. In their experiments, they coated a glass slide with molecules called "capture probes" that would grab onto microRNAs in their vicinity. Then, in different trials, they dropped onto the slide samples of solutions containing five different microRNAs. In one case, the solution that carried the microRNAs was human blood serum—the fluid component with the blood cells removed. To tell them RNA had been captured by one of the probes, they relied on a third type of molecule—fluorescent DNA strands that bind to the microRNA and emit light when they do. Only specific DNA sequences will bind to particular RNAs, so by varying the arrangement of the building blocks that make the DNA, the researchers engineered strands that would attach to the different microRNAs. What makes their method unique is that the DNA and RNA connect so weakly they don't stay stuck. DNA strings latch onto and detach from RNA in particular rhythms. When the researchers observe this through a super-sensitive fluorescence microscope, it looks like a firefly blinking. They can confirm the capture of different microRNAs based on the blink rate—its "kinetic fingerprint." Although microRNAs have been detected in blood serum before, this approach is more direct and suffers virtually no false positives. Source: University of Michigan

Biochemists solve 'address problem' in cells that leads to lethal kidney disease

 

---

Carla Koehler, UCLA professor of chemistry and biochemistry, is shown in her laboratory.

Research by UCLA biochemists may lead to a new treatment -- or even a cure -- for PH1, a rare and potentially deadly genetic kidney disease that afflicts children. Their findings also may provide important insights into treatments for Parkinson's disease, Alzheimer's disease and other degenerative diseases.

Led by Carla Koehler, a professor of chemistry and biochemistry in the UCLA College, the researchers identified a compound called dequalinium chloride, or DECA, that can prevent a metabolic enzyme from going to the wrong location within a cell. Ensuring that the enzyme -- called alanine: glyoxylate aminotransferase, or AGT -- goes to the proper "address" in the cell prevents PH1.

The findings were published online in the Proceedings of the National Academy of Sciences and will appear later in the journal's print edition.

In humans, AGT is supposed to go to an organelle inside the cell called the peroxisome, but for people with a particular genetic mutation, the enzyme mistakenly goes instead to the mitochondria -- tiny power generators in cells that burn food and produce most of the cells' energy -- which causes PH1.

Koehler's team demonstrated that adding small amounts of DECA, which is FDA-approved, to cells in a Petri dish prevents AGT from going to the mitochondria and sends it to its proper destination, the peroxisome.

"In many mutations that cause diseases, the enzyme doesn't work," Koehler said. "In PH1 the enzyme does work, but it goes to the wrong part of the cell. We wanted to use DECA in a cell model to block AGT from going to the wrong address and send it back to the right address. DECA blocks the mitochondria 'mailbox' and takes it to the peroxisome address instead."

How often did it work?

"All the time," said Koehler, a member of UCLA's Jonsson Comprehensive Cancer Center, Molecular Biology Institute and Brain Research Institute.

For people with the mutation, the correct peroxisome address is present in AGT, but it is ignored because it is accompanied by the address of the mitochondria, which the cell reads first, Koehler said.

Koehler, who also is a member of the scientific and medical advisory board of the United Mitochondrial Disease Foundation, hopes to find out whether a similar "correct address" strategy can slow cancer down. Her laboratory has identified approximately 100 other small molecules, which she calls MitoBloCKs, that she and her colleagues are testing for their ability to combat Parkinson's, Alzheimer's and other diseases.

PH1 -- short for primary hyperoxaluria 1 -- starts at birth and is usually fatal for patients who do not receive both kidney and liver transplants. Approximately half of those with the disease have kidney failure by age 15. Koehler has presented her findings to the Oxalosis and Hyperoxaluria Foundation, which provides support for PH1 patients and their families.

Scientists' ability to diagnose rare diseases has improved in recent years because technological advances in genomics have made it easier to identify more genetic mutations, Koehler said.

According to Koehler, to treat diseases, scientists must first understand how proteins like AGT move inside the cell. Her research, which encompasses biochemistry, genetics and cell biology, studies how mitochondria are assembled and function, how proteins enter the mitochondria and reach the right location inside cells, and how mitochondria communicate with the rest of the cell.

Her laboratory uses model systems that enable them to study the biochemistry in a way that is not possible with humans. Much of the work is conducted in yeast.

"It's exciting that our studies in baker's yeast, a typical laboratory model, might be able to help kids with a complicated disease," Koehler said.

---

Story Source:

The above story is based on materials provided by University of California - Los Angeles. The original article was written by Stuart Wolpert. Note: Materials may be edited for content and length.

---

Journal Reference:

1. N. Miyata, J. Steffen, M. E. Johnson, S. Fargue, C. J. Danpure, C. M. Koehler. Pharmacologic rescue of an enzyme-trafficking defect in primary hyperoxaluria 1. Proceedings of the National Academy of Sciences, 2014; DOI: 10.1073/pnas.1408401111

---