Action of Vessel Growth Inhibitor Pinned Down
Researchers at Children’s Hospital Boston have discovered the cellular target of angiostatin, the protein made famous a decade ago for its seemingly miraculous ability to cure cancer in mice. According to the paper, published online May 22 in Blood, angiostatin performs its myriad functions by interacting with proteins in mitochondria, small organelles that serve as the cell’s power generators.
Courtesy Kashi Javaherian
In these pictures of a human skin cancer cell treated with angiostatin, green (left) indicates the location of fluorescently labeled antibodies that specifically bind to angiostatin. Red fluorescence (middle) indicates the location of a special dye that targets mitochondria. An overlay of the two images (right) shows that angiostatin concentrates in the same place as mitochondria in the cell. The blue region is a fluorescent stain (DAPI) that targets the nucleus.
Angiostatin was first discovered in 1994 by the late Judah Folkman as an
anti-angiogenic protein in mice, blocking blood vessel formation in tumors
and effectively starving them into remission. Since then, researchers have
worked to elucidate angiostatin’s mechanism, hoping to learn enough
to reproduce the same antitumor effects in humans. Unfortunately, although
several proteins have been found to bind to angiostatin, a clear picture
of how it works has yet to emerge.
One problem may simply be that researchers have been looking in the wrong place. In the latest study, led by first author Tong-Young Lee, a postdoctoral fellow in the Vascular Biology Program at Children’s, the researchers found that angiostatin binds to mitochondrial malate dehydrogenase, a protein involved in the crucial metabolic process known as the Krebs cycle. This may come as a surprise to some scientists, because until now, there had been little evidence of angiostatin’s direct involvement in mitochondria.
“Eight years ago I came across malate dehydrogenase as a major binding protein of angiostatin,” recalled Kashi Javaherian, senior author of the study, an HMS lecturer on surgery and a senior research associate in the Vascular Biology Program. “But it was so far-fetched at the time, I didn’t touch it.”
Now, the idea no longer seems far-fetched. By treating a variety of cell types with fluorescently labeled angiostatin and observing them under the microscope, Lee found that whenever a cell took up the protein into its cytoplasm, the angiostatin would appear as a fluorescent signal in the mitochondria. Furthermore, when Lee applied short interfering RNA to some cells to dampen production of another protein, ATP synthase, the cells displayed significantly lower levels of mitochondrial angiostatin. This fact, combined with previous observations that angiostatin binds ATP synthase on the outer cell membrane, led Lee and Javaherian to think that ATP synthase is at least partially involved in the process of transporting angiostatin from outside the cell into the mitochondria.
According to the researchers, the challenge now is to find out exactly how angiostatin makes its way to the mitochondria and to explore the potential of delivering anticancer drugs to the organelles.
“We think that the action is not so much on the cell surface,” said Javaherian. “Sure, [angiostatin] has to bind to the cell surface in order to go inside, but the main action takes place in mitochondria.”
Students may contact Kashi Javaherian at Kashi.Javaherian@childrens.harvard.edu for more information.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: National Institutes of Health, National Aeronautics and Space Administration, Deutsche Forschungsgemeinschaft, Department of Defense, the Breast Cancer Research Foundation; the content of this work is the responsibility solely of the authors.
Study Lifts Mask off Latent Virus
Though the makers of the sixth Harry Potter movie downplayed it, the young wizard’s mysterious invisibility cloak gave him vitally important passage and power. The same holds true for the herpes simplex virus (HSV), other herpesviruses, and the human immunodeficiency virus (HIV), all of which can hide indefinitely inside cells in a dormant state. Safely cloaked, these viruses remain protected from antiviral medications yet at the same time poised to erupt back into infection.
It is not clear yet how the cloak forms—whether viruses mask themselves or if this latent state is part of an imperfect defense mounted by cells—but new work from David Knipe, the Higgins professor of microbiology and molecular genetics, has determined precisely which viral genes are responsible for weaving this molecular veil. The work may eventually lead to strategies that make latent viruses vulnerable to antiviral drugs by preventing latency or knocking viruses out of latency.
Though this study focused on herpes, new strategies may apply directly to HIV, which, according to an earlier report, uses a molecularly similar cloaking device. It may be even more feasible for HIV, said Knipe, because HIV viruses become latent in immune cells rather than in the less dispensable sensory neurons that herpes hides inside.
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Though this study focused on herpes, new strategies may apply directly to HIV, which, according to an earlier report, uses a molecularly similar cloaking device. |
When herpes viruses infect epithelial cells, they cause infection by mimicking cellular genes and replicating alongside them. A spool-like chromatin structure forms in the nucleus to assist in viral DNA replication. But when these viruses enter sensory neurons, they become latent. Rather than replicating, all of the viral DNA save an exclusive set of active latency-associated transcripts lurks quietly inside the cell. The chromatin structure that forms with the viral DNA contains modifications—changes to underlying proteins called histones—that make the assembly more compact. This tighter structure makes it impossible for the transcription factors that read genetic instructions to access the genes.
Knipe and first author Anna Cliffe, HMS research associate, discovered the exact histone modification that cloaks the DNA, trimethylation of the residue number 27 (lysine) on histone H3, or H3K27me3. They also found that this modification only emerges along with the latency-associated transcripts, suggesting that these bits of noncoding RNA promote this gene-silencing modification. The researchers, who will next try to understand how these molecules interact to weave the cloak, described this work in the August Journal of Virology.
Students may contact David Knipe at david_knipe@hms.harvard.edu for more information.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources:
The National Institutes of Health; the content of the work is the responsibility
solely of the authors.
ONLINE ONLY
Five Gene Variants Predict Lung Cancer Survival
A surge of genetic testing services has many people asking a difficult question: What is my risk of getting cancer? But for those just diagnosed, doctors must field a new question: How long do I have? For patients with non–small cell lung cancer, this question is particularly difficult to answer. Detected early, during stage I or II of the disease, patients have pretty good odds at five-year survival. But actual survival times vary widely and, for most patients, diagnosis does not come until the disease has reached stage III or IV.
New work from HSPH, however, has identified in the lung cancer tumor genome a set of five common genetic variations—called single nucleotide polymorphisms, or SNPs—that predict survival. Patients with stage I or II lung cancer and none of these risk-associated variations, called risk alleles, can expect to live five or more years. But for those with tumors that have three or more risk alleles, the odds drop precipitously to a 50–50 chance of surviving just 20 months.
For those with tumors having three or more risk alleles, the odds drop precipitously to a 50–50 chance of surviving just 20 months. |
The study’s authors, led by David Christiani, HSPH professor of occupational medicine and epidemiology and HMS professor of medicine, examined tumor tissue collected at Massachusetts General Hospital, sequenced the cells, and then validated the data against the Cancer Genome Atlas. In collaboration with colleagues from the HSPH Program in Quantitative Genomics, the researchers filtered out the genetic variations not statistically associated with survival. To further narrow the field, the team analyzed tumor tissue from Norwegian patients with Stage I or II lung cancer to identify the risk alleles present in the samples, assembled by the National Institute of Occupational Health in Oslo, Norway. This step trimmed the set to five risk-associated variants located within four known genes: STK39, PCDH7, A2BP1 and EYA2. The work is described in the June Journal of Clinical Oncology.
While these molecular markers have the potential to add accuracy to survival predictions made by the traditional, histological, cells-under-a-microscope stage classifications, it is unlikely that the staging system will incorporate such markers in the near term. Globally, genetic analyses remain out of reach for most clinics.
But for early-stage lung cancer, these markers could help clinicians stratify patients into different risk groups, said co-author Rebecca Heist, HMS associate professor of medicine at MGH. For instance, assuming these markers are validated in a larger study, a clinician might advise a patient with multiple risk alleles to endure the side effects of postsurgical chemotherapy, increasing the odds of long-term survival.
Students may contact David Christiani at dchris@hsph.harvard.edu for more information.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: The National Institutes of Health (National Cancer Institute)
and the Norwegian Cancer Society; the content of the work is the responsibility
solely of the authors.
Molecular Defect in Early-onset Alzheimer’s ID’d
Nine out of ten cases of inherited early-onset Alzheimer’s disease trace back to mutations in presenilin 1 or 2, proteins in neurons that help form and maintain memories. While mice with presenilin mutations follow the same progression as human Alzheimer’s patients, with memory loss, compromised ability to learn, and dementia, the exact molecular effects of these mutations have remained a mystery.
To solve this puzzle, Jie Shen, HMS associate professor of neurology at Brigham and Women’s Hospital, approached the problem with a painstaking genetic dissection of the workings of presenilin on both the sending and receiving sides of neural synapses, the junctions where signals are passed between nerve cells. She found that presenilin defects quash the release of neurotransmitters from presynaptic neurons, but have no effect on the transmission of synaptic signals in postsynaptic neurons.
This confluence of data suggests that diminished neurotransmitter flow may be a precursor to neurodegeneration, a hallmark of diseases such as Alzheimer’s and Parkinson’s. |
The discovery identifies the earliest disease-causing change in brain function caused by presenilin defects and identifies potential new therapeutic targets for early-onset Alzheimer’s.
The work dovetails with parallel findings from Shen’s and other labs showing that defects in four Parkinson’s disease genes similarly stifle the release of the neurotransmitter dopamine. This confluence of data suggests that diminished neurotransmitter flow may be a precursor to neurodegeneration, a hallmark of diseases such as Alzheimer’s and Parkinson’s.
Shen studied the presenilin mutations in neurons in the seat of memory in the brain, the hippocampus, using conditional double-knockout mice. The genetic knockouts precisely targeted CA3 (presynaptic) and CA1 (postsynaptic) pyramidal neurons with mutations that impaired their production of presenilins. She found that the loss of presenilins in presynaptic neurons stifles the Ryanodine Receptor (RyR), which gates calcium release from the endoplasmic reticulum. This disruption knocks down calcium efflux from the endoplasmic reticulum, causing a shortfall that compromises the release of the neurotransmitter glutamate. This interruption of the signal from one neuron to the next impairs long-term potentiation, a key neural mechanism for forming and maintaining memories. Molecules in this cascade could represent novel therapeutic drug targets for inherited forms of Alzheimer’s disease, said Shen, who described these new insights in the July 30 Nature.
Students may contact Jie Shen at jshen@rics.bwh.harvard.edu for more information.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: The National Institutes of Health; the content of the work is the responsibility solely of the authors.
Speed Dating: Matchmaking Algorithm Finds Kindred Genes
Over the last six years, the National Institutes of Health (NIH) library of publicly available genomic data has mushroomed. Researchers can now go online and peruse tens of thousands of datasets that scientists have deposited (the NIH requires that all government-funded genomic research be publicly available). But as data swells, so do the challenges: how do researchers interrogate this information torrent to find genes of interest? Forget any analogies of needles and haystacks; this is like trying to characterize every single stalk of hay from a Kansas prairie.
Now, reporting in the Aug. 6 issue of Cell Metabolism, scientists in the HMS Department of Systems Biology have developed a computational tool that can sift these massive datasets for genes of interest with remarkable speed, completing in days what normally takes one to three years. The group has also teamed up with researchers at Brigham and Women’s Hospital to verify the tool’s findings.
“We’re living in the postgenomic era now where out of 20,000 protein-coding genes only about 5,000 of them are really well studied,” said Vamsi Mootha, an HMS associate professor of systems biology and an HMS associate professor of medicine at Massachusetts General Hospital. Along with Barry Paw, HMS assistant professor of medicine at Brigham and Women’s Hospital, Mootha is co-senior author on the paper. “Now we have the opportunity to look more deeply into what the other 15,000 do,” he said.
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“We’re living in the postgenomic era now where out of 20,000 protein-coding genes only about 5,000 of them are really well studied. Now we have the opportunity to look more deeply into what the other 15,000 do.” —Vamsi Mootha |
According to Mootha and postdoctoral scientist Roland Nilsson, the vast aggregate of genomic data in the NIH depository is like a network of pools. Each pool is an individual dataset, and each dataset can contain results from hundreds of microarray experiments. Each pool, then, is swimming with countless bits of genomic clues. The trick is fishing out the particular bits you need.
To do this, Nilsson developed an algorithm that can mine this depository quickly. Starting from a handful of known genes, the algorithm hunts through all datasets and finds every gene that behaves like the initial ones.
“It’s like fishing,” said Mootha. “The initial gene set, that’s your bait. You then cast it into each pond, one at a time, and see what you catch.”
The team tested the algorithm on a series of eight genes known to be essential for hemoglobin synthesis, and fished out five genes that had never before been associated with blood production. The group then teamed up with Paw, a pediatric hematologist-oncologist, to test these findings in zebrafish. Paw and postdoctoral scientist Iman Schultz confirmed in animal models that these five genes were, in fact, essential for hemoglobin production.
“Using traditional methods, it could take well over a year to identify one of these genes,” said Paw, “whereas here, through the combined work, we can do a really quick, genomewide screen and then follow up with validation in an experimental model in a few weeks.”
Students may contact Vamsi Mootha (vamsi@hms.harvard.edu) or
Barry Paw (bpaw@rics.bwh.harvard.edu) for
more information.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: The National Institutes of Health, the March of Dimes Foundation, the American Diabetes Association/Smith Family Foundation, and the Howard Hughes Medical Institute; the content of this work is the responsibility solely of the authors.