Cell's power generator depends on long-sought protein: 50-year search for calcium channel ends

Mitochondria, those battery-pack organelles that fuel the energy of almost every living cell, have an insatiable appetite for calcium. Whether in a dish or a living organism, the mitochondria of most organisms eagerly absorb this chemical compound. Because calcium levels link to many essential biological processes—not to mention conditions such as neurological disease and diabetes—scientists have been working for half a century to identify the molecular pathway that enables these processes.

After decades of failed effort that relied on classic biochemistry and membrane protein purification, Vamsi Mootha, HMS associate professor of systems biology, and colleagues have discovered, through a combination of digital database mining and laboratory assays, the linchpin protein that drives mitochondria's calcium machinery.

"This channel has been studied extensively using physiology and biophysics, yet its molecular identity has remained elusive," said Mootha, who also has appointments at Massachusetts General Hospital and at Broad Institute. "But thanks to the Human Genome Project, freely downloadable genomic databases, and a few tricks -- we were able to get to the bottom of it."

These findings will appear online June 19 in Nature.

The results build on work from Vamsi and his group over the past decade. In 2008, he and his team published a near-comprehensive protein inventory, or proteome, of human and mouse mitochondria. This inventory, called MitoCarta, consisted of just over 1,000 proteins, most of which had no known function.

In a September 2010 paper, Mootha's group described using the MitoCarta inventory to identify the first protein specifically required for mitochondrial calcium uptake. Their strategy was simple. They knew that mitochondria from humans and Trypanosomes (a parasitical organism), but not baker's yeast, are capable of absorbing large amounts of calcium. By simply overlapping the mitochondrial protein profiles of these three organisms, the group could spotlight roughly 50 proteins out of the 1,000 that might be involved with calcium channeling. They found that one protein, which they dubbed MICU1, is essential for calcium uptake.

"That was an significant advance for the field," says Mootha. "We showed that MICU1 was required for calcium uptake, but because it did not span the membrane, we doubted it was the central component of the channel. But what it provided us with was live bait to then go and find the bigger fish."

Traditionally, researchers used standard laboratory methods for such a fishing exhibition, such as attaching biochemical hooks to the protein, casting it into the cell's cytoplasm, then reeling it back in the hope that another, related protein will have bitten. But MICU1's function as a regulator of a membrane channel made this technically prohibitive. Instead, graduate student Joshua Baughman and postdoctoral researcher Fabiana Perocchi went fishing in publicly available genomic databases.

With MICU1 as their point of reference, they scoured those databases that measure whole genome RNA and protein expression, as well as an additional database containing genomic information for 500 species, and looked for proteins whose activity profile mirrored MICU1's. A single anonymous protein with no known function stood out. The researchers named it MCU, short for "mitochondrial calcium uniporter."

To confirm that MCU is central to mitochondria's calcium absorption, the team collaborated with Alnylam Pharmaceuticals, a company that leverages a laboratory tool called RNAi in order to selectively knock out genes in both cells and live animals. Using one of the company's platforms, the researchers deactivated MCU in the livers of mice. While the mice displayed no immediate reaction, the mitochondria in their liver tissue lost the capacity to absorb calcium.

This basic science finding may prove relevant in certain human diseases. "We've known for decades now that neurons in the brains of people suffering from neurodegenerative disease are often marked by mitochondrial calcium overload," said Mootha, an expert on rare mitochondrial diseases who sees patients at Massachusetts General Hospital when he's not in the lab.

"We also know that the secretion of many hormones, like insulin, are triggered by calcium spikes in the cell's cytoplasm. By clearing cytosolic calcium, mitochondria can shape these signals. Scientists studying the nexus of energy metabolism and cellular signaling will be particularly interested in MICU1 and MCU. It's still very early, but they could prove to be valuable drug targets for a variety of diseases – ranging from ischemic injury and neurodegeneration to diabetes."

More information: "Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter" Nature, online publication June 19, 2011.

Provided by Harvard Medical School (news : web)

Turning off cancer's growth signals

 

One hallmark of cancer cells is uncontrollable growth, provoked by inappropriate signals that instruct the cells to keep dividing. Researchers at MIT and Brigham and Women’s Hospital have now identified a new way to shut off one of the proteins that spreads those signals — a receptor known as HER3.

Drugs that interfere with HER3’s better-known cousins, EGFR and HER2, have already proven effective in treating many types of cancer, and early-stage clinical trials are underway with antibodies directed against HER3. HER3 is of great interest to cancer biologists because it is commonly involved in two of the deadliest forms of the disease, ovarian and pancreatic cancer, says MIT Professor Linda Griffith, who led the research team with Harvard Stem Cell Institute and Brigham and Women’s cardiologist Richard Lee.

The study, published online May 26 in the Journal of Biological Chemistry, resulted from a serendipitous finding in a regenerative-medicine project. Co-first author Luis Alvarez, who earned his PhD from MIT during a three-year leave from the Army, was interested in regenerative medicine because he knew many soldiers who had been wounded in Iraq and Afghanistan.

While looking for ways to promote bone regrowth, Alvarez developed a series of paired proteins that the researchers thought might promote interactions between growth receptors such as HER3 and EGFR to control growth and differentiation.

Alvarez’s proteins had some impact on regeneration, but the researchers also noticed that in some cases, they appeared to shut off cell growth and migration. Alvarez and others in Griffith’s lab decided to see what would happen if they treated cancer cells with the protein. To their surprise, they found that the cells stopping growing, and in some cases died.

“It was not something we were expecting to see — you don’t expect to shut off a receptor with something that normally activates it — but in retrospect it seemed obvious to try this approach for HER3,” says Griffith, the School of Engineering Professor of Innovative Teaching in MIT’s Department of Biological Engineering and director of the Center for Gynepathology Research. “We pursued it only because we had people in the lab working with cancer cells, and we thought, ‘Since it had these effects in stem cells, let’s just try this in tumor cells, and see if something interesting happens.’”

Targeting vulnerability

Around the same time, Griffith developed a personal interest in this family of cell receptors: She was diagnosed with a form of breast cancer that often overexpresses the receptor EGFR.

EGFR has received much attention from biologists — the cancer drugs Erbitux, Iressa and Tarceva all target it — but not all cancers that overexpress the EGFR respond to targeted therapies. The first highly successful targeted chemotherapy, Herceptin, goes after another member of the family, the HER2 receptor.

The new MIT protein targets a specific vulnerability of HER3: To convey its growth-stimulating signals to the rest of the cell, HER3 must pair up with another receptor, usually HER2.

The new protein, which consists of a fused pair of neuregulin molecules, disrupts that pairing. Single molecules of neuregulin normally stimulate the HER3 receptor, promoting cell growth and differentiation. However, when the paired neuregulin is given to cells, it binds together two adjacent HER3 receptors, preventing them from interacting with the HER2 receptors they need to send their signals.

The researchers tested the molecule in six different types of cancer cells that overexpress HER3, and found that it effectively shut off growth in all of them, including a cell type that is resistant to drugs that target EGFR.

Mark Moasser, a professor of oncology at the University of California at San Francisco, described the new technique as clever and elegant, adding that more experiments are needed to determine if it will be effective in living organisms. “Based on the mechanism, it has potential, and it lays the groundwork for a lot of future work,” says Moasser, who was not involved in this study.

The MIT and Brigham and Women’s team is now working on a new version of the molecule that would be more suited to tests in living animals. They plan to undertake such testing soon under the leadership of Steven Jay, a joint MIT/Brigham and Women’s postdoc and co-first author of the new paper. MIT postdoc Elma Kurtagic and graduate student Seymour de Picciotto are also first authors of the paper.
This story is republished courtesy of MIT News (http://web.mit.edu/newsoffice/), a popular site that covers news about MIT research, innovation and teaching.

More information: Paper online: http://www.jbc.org … 093.abstract

Provided by Massachusetts Institute of Technology (news : web)

Scientists discover new component of key growth-regulating signaling pathway

Researchers in the lab of Whitehead Institute Member David Sabatini have identified a new substrate of the mammalian target of rapamycin (mTOR) kinase, called Grb10, by using a two-pronged approach of mass spectrometry and kinase specificity profiling.

“These results show that mTOR participates in most key cellular processes, consisting with its established role in common diseases like diabetes, cancer, and neurodegeneration” says Sabatini, who is also a Howard Hughes Medical Institute (HHMI) investigator and a professor of biology at MIT.

The research is published in the June 10 issue of Science.

The Grb10 protein was known to interact with insulin receptors and to inhibit the ability of cells to respond to insulin. By identifying the relationship between Grb10 and mTOR, Peggy Hsu, a former graduate student in the Sabatini lab and first author of the Science paper, was able to show that Grb10 is important for mTOR to inhibit signaling downstream of extracellular growth factors like insulin. This provides researchers with a more detailed understanding of the function of mTOR, especially in the context of cancer, and opens up new areas for mTOR research.

“These results show that mTOR participates in most key cellular processes, consisting with its established role in common diseases like diabetes, cancer, and neurodegeneration” says Whitehead Member David Sabatini.

The role of mTOR in nutrient sensing and regulation of cell growth has been conserved from yeast to worms, flies, and mice.  In humans, it is also important in overall organismal nutrient metabolism and organism size; dysregulation of mTOR has been linked to diabetes and some cancers. Despite its biological and medical importance—drugs that inhibit mTOR are used to treat certain cancers and to suppress the immune system to prevent transplant rejections—little is actually known about the substrates that mTOR targets or the precise manner in which it affects multiple cellular processes.

Hsu, who is now finishing her medical degree at Harvard Medical School, says that until recently, the search for mTOR substrates has been non-systematic and hamstrung by rapamycin’s limited inhibition of mTOR. With the advent of new mTOR inhibitors that target mTOR directly, including the inhibitor Torin1 that Hsu used in her work, researchers are now able to get a more complete picture of what mTOR regulates.

According to Hsu, use of these new mTOR inhibitors in conjuction with mass spectrometry and the kinase specificity profiling method will transform both future and current mTOR research.

“I think this will open up different areas of potential exploration,” says Hsu, who worked closely with the labs of Michael Yaffe at MIT and Jarrod Marto at Harvard Medical School. “I hope this work allows other reseachers to make a connection very quickly by looking through our data, and to basically say, ‘Aha! I thought mTOR would be involved in process X. And now maybe I have a way to study it.’ ”

More information: “The mTOR-Regulated Phosphoproteome Reveals a Mechanism of mTORC1-Mediated Inhibition of Growth Factor Signaling” Science, June 10, 2011

Provided by Whitehead Institute for Biomedical Research (news : web)

Researchers find new clues about protein linked to Parkinson's disease

Researchers at the Keck School of Medicine of the University of Southern California (USC) have uncovered structural clues about the protein linked to Parkinson's disease (PD), which ultimately could lead to finding a cure for the degenerative neurological disorder.

The alpha-synuclein (?-synuclein) protein is commonly found in the healthy human brain even though its function is not clear. The protein has been the subject of substantial Parkinson's research, however, because it is a major component in the protein clumps found in PD cases.

Unlike most proteins, which are typically rigid and occur in one definitive form, the alpha-synuclein protein can fold and change its structure. Researchers Tobias S. Ulmer, Ph.D. and Sowmya Bekshe Lokappa, Ph.D. at the Keck School-affiliated Zilkha Neurogenetic Institute have determined that the energy difference between two particular alpha-synuclein structures is less than previously speculated.

Their study, to be published in the June 17 issue of The Journal of Biological Chemistry, is the first to quantify that energy difference, 1.2±0.4 kcal/mol.

"We're trying to understand the mechanisms of protein folding and misfolding," said Ulmer, the study's principal investigator and an assistant professor in the Department of Biochemistry and Molecular Biology at the Zilkha Neurogenetic Institute. "Then we can say why something is going wrong, which is essential to treating neurodegenerative disorders like Parkinson's."

If proteins misfold, they are repaired or they break down. However, when alpha-synuclein misfolds it aggregates and becomes toxic to surrounding nerve cells, Ulmer said. Understanding its folding and finding what causes aberrant folding is therefore key to determining the root cause of the disorder, he added.

To put the discovery into perspective, Ulmer compared the energy that researchers thought was needed to change the protein's structure to hurricane-force winds and the actual energy required to a light summer breeze. The experiments were conducted in 2010, measuring the energy of elongated and broken helix forms of alpha-synuclein through circular dichroism spectroscopy, fluorescence spectroscopy and isothermal titration calorimetry.

"There may be a continuous interconversion between folded alpha-synuclein structural states that might contribute to its pathological misfolding," said Lokappa, a post-doctoral research associate at the Center for Craniofacial Molecular Biology at USC and the study's co-author. "But we need to have even better insight into the mechanisms of protein folding and misfolding to explain what's going wrong in the brain."

The paper is the sixth in a series of studies that Ulmer has published on alpha-synuclein.

Parkinson's is a neurological disorder that has no cure or determined cause. It is a slow-progressing degenerative disease that most commonly affects motor function. According to the National Parkinson Foundation, the disorder is the second-most common neurodegenerative disease after Alzheimer's, affecting 1 million people in the United States and some 4 million worldwide.

More information: http://www.jbc.org … 4/21450.full

Provided by University of Southern California (news : web)