Structure of Channel in Human Mitochondria Reveals New Puzzles

It is a rare event when a long-studied protein yields its structure to new experimental methods, and rarer still when that structure surprises the experts and intrigues even the casual observer. Such is the case for the recent model of a human voltage-dependent anion channel (VDAC-1), solved by nuclear magnetic resonance in the lab of Gerhard Wagner and reported in the Aug. 29 Science.

VDAC-1 allows passage of ions and small molecules including ATP across the mitochondrial outer membrane. It is also involved in controlling release of mitochondrial proteins that lead to programmed cell death. Wagner, the Elkan Blout professor of biological chemistry and molecular pharmacology at HMS, and his laboratory began working on the channel because, he said, “I realized that the most crucial events in apoptosis happen at the mitochondrial membrane.” Dysregulation of apoptosis can cause cancer and other disorders.

Courtesy Gerhard Wagner

Odd structure discovered. This human VDAC-1 solution structure shows an odd number (19) of antiparallel beta strands, with the two terminal strands (dark gray) meeting in a parallel orientation. Like beta barrels with an even number of strands, both termini still protrude from the same side of the barrel, thanks to a 23-amino acid N-terminal tail (pink) that reaches through the channel and is involved in voltage gating.

Human VDAC-1 joins 32 prokaryotic beta barrel membrane proteins whose structures have been solved. The beta strands are antiparallel, and all known beta barrels have an even number of strands. According to lead author Sebastian Hiller, a research fellow in Wagner’s lab, it was widely believed that all beta barrels—including eukaryotic ones—would display an even number of strands, since an odd number would make the terminal strands parallel, a less stable orientation.

The team studied recombinant VDAC-1 refolded in detergent balls called micelles, a system established by former graduate student Thomas Malia. Determining the protein’s fold involved assigning more than 600 nuclear Overhauser effects (NOEs) between atomic nuclei to define their spatial proximity and therefore the overall topology of the protein. Initially, Hiller and Wagner thought VDAC-1 fit the mold with an even number of strands, but suddenly, each independently realized that the data were telling them there were 19 strands. “Gerhard said, ‘It must have 19,’” Hiller recalled, “and I had just found two new NOEs,” which convinced them.

One striking feature of the structure is its ability to account for VDAC-1’s ion preferences: one negative and two positive patches are seen, and VDAC-1 displays a 2:1 preference for anions. An experiment with detergent carrying a paramagnetic spin label identified a belt of amino acid residues contacting detergent. Interaction sites with binding partners such as the anti-apoptotic protein Bcl-x(L) were also observed.
Hiller and Wagner expect that the topology in the micelles is the same in lipid bilayers. “We are not aware of any structure where a membrane protein adopts different structures in different environments,” Wagner said. Nonetheless, they are keen to take on the challenge of studying the protein in a bilayer.

Despite VDAC-1’s odd number of beta strands, both termini still lie on the same side of the channel, thanks to a long N-terminal tail that reaches through it. “We think this part is crucial for gating,” Wagner said. The group has begun a collaboration to study by computational modeling how gating is accomplished.

In fact, Hiller said, “we have a list of 30 or 40 things that I would like to do.”

—Megan Talkington

Conflict Disclosure: The authors declare no conflicts of interest.

Funding Sources: The National Institutes of Health; the Swiss National Science Foundation; the Wenner-Gren Foundation, Stockholm; and the Ludwig Foundation for Cancer Research

Value of Direct-to-consumer Drug Advertising May Be Oversold

Direct-to-consumer advertising may not be giving big pharma such a big bang for its buck, after all. Despite the billions spent on bringing drug marketing campaigns straight into patients’ living rooms, such strategies have a modest effect at best—and in some cases, no effect at all.

“People tend to think that if direct-to-consumer advertising wasn’t effective, pharma wouldn’t be doing it,” said HMS professor of ambulatory care and prevention Stephen Soumerai, principal investigator on the study. “But as it turns out, decisions to market directly to consumers are based on scant data.”

The research was based in the Department of Ambulatory Care and Prevention of HMS and Harvard Pilgrim Health Care and appeared online Sept. 2 in BMJ. It is the first-ever controlled study of direct-to-consumer advertising (DTCA) of pharmaceuticals.

Currently, the United States and New Zealand are the only countries that allow drug companies to advertise directly to patients, and, as of 2005, pharma was spending about $5 billion annually in the United States on such campaigns.

The researchers took advantage of the proximity of the U.S. to Canada to conduct their study. DTCA is illegal across the border. Not surprisingly, however, national borders are leaky and American media regularly crosses over. As a result, Canadians, like Americans, are swamped with these ads—with one key exception.

All American advertisements are in English. Yet Canada has a significant French-speaking population. As a result, residents of Quebec, on the whole, are far less exposed to DTCA than other Canadians. The researchers compared prescription rates for advertised drugs in English-speaking Canadian provinces with rates in Quebec, where residents were purportedly less exposed to those same ads.

“It’s not an absolutely perfect control group,” said Michael Law, first author on the paper. “But as control groups go for this sort of observational study, it’s about as good as you get.”

Using information from IMS Health Canada, a health information company that receives data from a panel of about 2,700 Canadian pharmacies, the researchers analyzed prescription statistics over five years for each of three drugs: Enbrel (rheumatoid arthritis), Nasonex (nasal allergies), and Zelnorm (irritable bowel syndrome).

They found that for two of the drugs, Enbrel and Nasonex, DTCA had no effect whatsoever. Sales for Zelnorm, however, did spike noticeably in English-speaking Canada as soon as the ad campaign began. While prescriptions for the drug increased by over 40 percent, this jump was relatively short-lived, and after a few years, prescription rates in both groups resumed identical patterns.

The researchers hypothesize that DTCA may not be as effective as other types of consumer advertising because consumers cannot simply go out and buy prescription drugs.

—David Cameron

Conflict Disclosure: The authors declare no conflicts of interest.

Funding Sources: HMS, Harvard Pilgrim Health Care, The Social Sciences and Humanities Research Council of Canada, The Alberta Heritage Foundation for Medical Research, and the Agency for Healthcare Research and Quality

Peripheral Circadian Clocks Take Center Stage in Homeostasis

Charles Weitz found himself in an enviable position in 2002, following the discovery of functional, autonomous circadian clocks in peripheral tissue cells. The Robert Henry Pfeiffer professor of neurobiology possessed the tools to test two equally exciting, yet opposing hypotheses about the role of these pacemakers, which reside in a variety of tissues.

Researchers in the field wondered—do the clocks, which generate transcriptional rhythms, regulate the same genes everywhere to ensure that core physiological processes take place in parallel? Or do the clocks regulate genes in a tissue-specific manner, coordinating heterogeneous processes throughout the body?

“Usually, investigators hope for a particular experimental outcome,” explained Weitz. “We were so ignorant as a field, however, that either explanation for the abundance of clocks seemed appealing and informative.”

Weitz and colleagues compared the transcriptional profiles of liver and heart tissue at different times of the day and found little overlap between the two. Although circadian clocks share the same basic machinery in both tissues, they appear to direct different genes and physiological programs. But Weitz wanted to move beyond correlation.
Working with postdoctoral researchers Katja Lamia and Kai-Florian Storch, he demonstrated that the liver clocks contribute to homeostasis by triggering the release of stored glucose from hepatocytes during the resting phase in mice. The study appeared online Sept. 8 in Proceedings of the National Academy of Sciences. The new data support—and possibly extend—the leading hypothesis about the preponderance of peripheral pacemakers.

“Glucose levels need to be kept in a narrow range all the time, and we showed that the liver clocks keep mice from becoming hypoglycemic when they’re not ingesting food,” explained Weitz.

“This study indicates that you need the liver clocks to counterbalance the brain clock,” said professor Ueli Schibler of the University of Geneva, who wrote a commentary on the paper, which will appear in the Sept. 30 issue of PNAS. The brain clock, located in the suprachiasmatic nucleus, drives our fasting–feeding cycle and thus regulates sugar ingestion.

The Weitz lab focused on the liver clock after reviewing the transcriptional profiles from the original experiment. Many of the genes involved with glucose metabolism in hepatocytes—including Glut2, G6pt1 and Gck—displayed beautiful circadian rhythms.
To rule out the possibility that these genes are slaves to the master brain clock, the team disabled Bmal1—a core component of the circadian clock—in hepatocytes alone. Bmal1 remained active in the brain clock and other peripheral clocks. The team combined a conditional Bmal1 allele with a Cre recombinase transgene under the control of an albumin promoter to achieve the hepatocyte-specific effect.

The resulting mice displayed hypoglycemia during their resting phase, as derelict hepatocytes kept stored glucose to themselves, despite a systemic need for sugar. Blood sugar levels returned to normal when the mice resumed eating during their active phase. Glut2, G6pt1 and Gck rhythms disappeared.

“This suggests that the liver clock contributes to systemic homeostasis by anticipating the glucose-level drop that accompanies fasting,” said first author Katja Lamia, who is now at the Salk Institute for Biological Studies.

The fasting–feeding cycle allows animals to anticipate when nutrients will become available, making physiologic processes more efficient. But it also poses homeostatic challenges. The liver clock overcomes one of these.

“Perhaps part of the function of peripheral clocks is to undo unwanted side effects of the fasting–feeding cycle,” said Weitz.

Schibler cautions against assuming that all peripheral clocks keep the brain clock from wreaking homeostatic havoc. He said that they seem to serve a variety of functions. “But the counterbalancing concept is original and interesting.”

—Alyssa Kneller

Conflict Disclosure: The authors declare no conflicts of interest.

Funding Sources: National Institutes of Health. Katja Lamia is a Merck Fellow of the Life Sciences Research Foundation

Deletion Upstream of Autophagy Gene Linked to Crohn’s Disease

Since the release of the human haplotype map, or HapMap, genomewide association studies have uncovered a plethora of susceptibility loci for diseases from asthma to diabetes. But as noted by Mark Daly, HMS assistant professor of medicine at Massachusetts General Hospital, when it comes to association studies, “We don’t get biological insight just from a highly significant p value.”

“The key steps after discovering an association,” Daly said, “are to identify the precise causal variant, identify how it influences the action of a gene, and then connect that molecular function to a biological process that influences disease.”

Aiming to take these key steps and identify how a particular susceptibility locus affects disease risk, Daly began collaborating with gastroenterologist Ramnik Xavier, also an HMS assistant professor of medicine at MGH, to study the genetic component of Crohn’s disease. Characterized by inflammation of the digestive tract, Crohn’s is known to have a substantial genetic component and more than 30 loci have been linked to it. Despite these genetic clues, the molecules and pathways underlying disease pathogenesis have proven difficult to pin down.

Reporting in the September issue of Nature Genetics, co–senior authors Xavier and Daly, who is also affiliated with the Broad Institute, and colleagues describe a polymorphism upstream of a gene called IRGM that is the likely causal variant behind a previously identified SNP association. Variation within the coding region of IRGM, a poorly understood autophagy-related gene, had been excluded by previous studies, so the researchers had to look beyond the gene itself.

Using a new genotyping method that more finely parses the DNA, Steven McCarroll, a postdoctoral fellow working with Daly, identified a 20kb deletion that was highly correlated with the known SNP and with Crohn’s disease risk. Previously, these smaller copy number variants were nearly impossible to detect, but McCarroll spearheaded the development of a hybrid array that simultaneously analyzes SNPs and copy number variants; this technology accelerated his search for polymorphisms. The development of this array technology, published separately, appeared online Sept. 7, also in Nature Genetics.

As one might expect given its location upstream of the IRGM gene, the deletion polymorphism affects IRGM expression. But the mutation did not simply produce a “crippled promoter and reduced expression,” according to McCarroll, lead author of the study. Instead the researchers saw that the deletion actually stimulated IRGM expression in some cell types, such as primary smooth muscle cells, while suppressing it in others, including lymphoblastoid cells. These results suggest that the overall pattern of IRGM expression in individuals carrying the deletion, who are at higher risk of developing Crohn’s, is different from the pattern in individuals without the deletion, even though the IRGM protein is identical in both populations.

To determine how IRGM expression levels affect cellular function, the researchers reduced IRGM expression in cells using short interfering RNA. They saw that IRGM-deficient cells were impaired in their ability to destroy via autophagy potentially pathogenic bacteria. Specifically, when challenged with Salmonella, IRGM-deficient cells failed to efficiently encapsulate the bacteria in autophagic vesicles, the first step toward digesting invaders in the lysosome. Conversely, the bacterial load within autophagic vesicles increased with increasing levels of exogenous IRGM.

The possibility that deficits in autophagy might contribute to the pathogenesis of Crohn’s disease was first recognized when susceptibility loci were identified in or near two different autophagy-related genes, IRGM and ATG16L1. “Now,” said Daly, “genetics has given us hard evidence that the inherited susceptibility … of the disease to some degree resides in genes … that directly influence the front-line response to bacteria.”

—Kelly Dakin

Conflict Disclosure: The authors declare no conflicts of interest.

Funding Sources: The National Institute of Allergy and Infectious Diseases; Center for Computational and Integrative Biology, MGH; Lilly; the National Institute of Diabetes and Digestive and Kidney Diseases; the Burroughs Wellcome Fund; and the Crohn's and Colitis Foundation of America

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