Biochemists Solve the Structure of Cell's DNA Gatekeeper

Caltech scientists have produced the most detailed map yet of the massive protein machine that controls access to the DNA-containing heart of the cell.

In a new study, a team led by André Hoelz, an assistant professor of biochemistry, reports the successful mapping of the structure of the symmetric core of the nuclear pore complex (NPC), a cellular gatekeeper that determines what molecules can enter and exit the nucleus, where a cell's genetic information is stored.

The study appears in the April 15, 2016 issue of the journal Science, featured on the cover.

The findings are the culmination of more than a decade of work by Hoelz's research group and could lead to new classes of medicine against viruses that subvert the NPC in order to hijack infected cells and that could treat various diseases associated with NPC dysfunction.

"The methods that we have been developing for the last 12 years open the door for tackling other large and flexible structures like this," says Hoelz. "The cell is full of such machineries but they have resisted structural characterization at the atomic level."

The NPC is one of the largest and most complex structures inside the cells of eukaryotes, the group of organisms that includes humans and other mammals, and it is vital for the survival of cells. It is composed of approximately 10 million atoms that together form the symmetric core as well as surrounding asymmetric structures that attach to other cellular machineries. The NPC has about 50 times the number of atoms as the ribosome—a large cellular component whose structure was not solved until the year 2000. Because the NPC is so big, it jiggles like a large block of gelatin, and this constant motion makes it difficult to get a clear snapshot of its structure.

In 2004, Hoelz laid out an ambitious plan for mapping the structure of the NPC: Rather than trying to image the entire assembly at once, he and his group would determine the crystal structures of each of its 34 protein subunits and then piece them together like a three-dimensional jigsaw puzzle. "A lot of people told us we were really crazy, that it would never work, and that it could not be done," Hoelz says.

Last year, the team published two papers in Science that detailed the structures of key pieces of the NPC's inner and outer rings, which are the two primary components of the NPC's symmetric core. The donut-shaped core is embedded in the nuclear envelope, a double membrane that surrounds the nucleus, creating a selective barrier for molecules entering and leaving the nucleus.

By being able to piece these crystal structures into a reconstruction of the intact human NPC obtained through a technique called electron cryotomography—in which entire isolated nuclei are instantaneously frozen, with all of their structures and molecules locked into place, and then probed with a transmission electron microscope to produce 2-D images that can be reassembled into a 3-D structure—"we bridged for the first time the resolution gap between low-resolution electron microscopy reconstructions that provide overall shape and high-resolution crystal structures that provide the precise positioning of all atoms," Hoelz says.

With these structures known, the mapping of the rest of the NPC's symmetric core came quickly. "It is just like when solving a puzzle," he says. "By placing the first piece confidently, we knew that we would eventually be able to place all of them."

As described in the new paper, Hoelz's research group now has solved the crystal structures of the last remaining components of the symmetric core's inner ring and determined where all of the rings' pieces fit in the NPC's overall structure.

To do this, the team had to first generate a complete "biochemical interaction map" of the entire symmetric core. Akin to a blueprint, this map describes the interconnections and interactions of all of the proteins, as part of a larger cellular machine. The process involved genetically modifying bacteria to produce purified samples of each of the 19 different protein subunits of the NPC's symmetric core and then combining the fragments two at a time inside a test tube to see which adhered to each other.

The team then used the completed interaction map as a guide for identifying the inner ring's key proteins and employed X-ray crystallography to determine the size, shape, and position of all of their atoms. X-ray crystallography involves shining X-rays on a crystallized sample and analyzing the pattern of rays reflected off the atoms in the crystal. The team analyzed thousands of samples at Caltech's Molecular Observatory—a facility developed with support from the Gordon and Betty Moore Foundation that includes an automated X-ray beam line at the Stanford Synchrotron Radiation Laboratory that can be controlled remotely from Caltech—and the GM/CA beam line at the Advanced Photon Source at the Argonne National Laboratory.

"We now had a clear picture of what the key jigsaw pieces of the NPC looked like and how they fit together," says Daniel Lin, a graduate student in Hoelz's lab and one of two first authors on the study.

The next step was to determine how the individual pieces fit into the larger puzzle of the NPC's overall structure. To do this, the team took advantage of an electron microscopy reconstruction of the entire human NPC published in 2015 by Martin Beck's group at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. The images from Beck's group were relatively low resolution and revealed only a rough approximation of the NPC's shape, but they still provided a critical framework onto which Hoelz's team could overlay their atomic high-resolution images, captured using X-ray crystallography. The NPC is the largest cellular structure ever pieced together using such an approach.

"We were able to use the biochemical interaction map we created to solve the puzzle in an unbiased way," Hoelz says. "This not only ensured that our pieces fit in the electron microscopy reconstruction, but that they also fit together in a way that made sense in the context of the interaction map."

Hoelz said his team is committed to solving the remaining asymmetric parts of the NPC, which include filamentous structures that serve as docking sites for so-called transport factors that ferry molecules safely through the pore and other cellular machineries that are critical for the flow of genetic information from DNA to RNA to protein.

"I suspect that things are going to move very quickly now," Hoelz says. "We know exactly what we need to do. It's like we're climbing Mount Everest for the first time, and we've made it to Camp 4. All that's left is the sprint to the summit."

Along with Hoelz and Lin, additional Caltech authors on the paper, "Architecture of the symmetric core of the nuclear pore," include research technician Emily Rundlet; Thibaud Perriches, George Mobbs, and Karsten Thierbach, all postdoctoral scholars in chemistry working in the Hoelz lab; and graduate students Ferdinand Huber and Leslie Collins. Other coauthors on the paper include former Hoelz lab member Tobias Stuwe—the second cofirst author of the paper—as well as former lab members Sandra Schilbach, Yanbin Fan, Andrew Davenport (PhD '15), and Young Jeon.

The work was supported by the National Institute of General Medical Sciences; the Caltech-Amgen Research Collaboration; the German Research Foundation; the Boehringer Ingelheim Fonds; the China Scholarship Council; Caltech startup funds; an Albert Wyrick V Scholar Award from the V Foundation for Cancer Research; a Mallinckrodt Scholar Award from the Edward Mallinckrodt Jr. Foundation; a Kimmel Scholar Award from the Sidney Kimmel Foundation; and a Camille Dreyfus Teacher-Scholar Award from the Camille & Henry Dreyfus Foundation. Hoelz is also an inaugural Heritage Principal Investigator of the Heritage Medical Research Institute for the Advancement of Medicine and Science at Caltech.

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The detailed map is the first to determine the structure of a massive protein machine with near-atomic resolution.
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Nanoparticle-Based Cancer Therapies Shown to Work in Humans

A team of researchers led by Caltech scientists has shown that nanoparticles can function to target tumors while avoiding adjacent healthy tissue in human cancer patients.

"Our work shows that this specificity, as previously demonstrated in preclinical animal studies, can in fact occur in humans," says study leader Mark E. Davis, the Warren and Katharine Schlinger Professor of Chemical Engineering at Caltech. "The ability to target tumors is one of the primary reasons for using nanoparticles as therapeutics to treat solid tumors."

The findings, published online the week of March 21 in the journal Proceedings of the National Academy of Sciences, demonstrate that nanoparticle-based therapies can act as a "precision medicine" for targeting tumors while leaving healthy tissue intact.

In the study, Davis and his colleagues examined gastric tumors from nine human patients both before and after infusion with a drug—camptothecin—that was chemically bound to nanoparticles about 30 nanometers in size.

"Our nanoparticles are so small that if one were to increase the size to that of a soccer ball, the increase in size would be on the same order as going from a soccer ball to the planet Earth," says Davis, who is also a member of the City of Hope Comprehensive Cancer Center in Duarte, California, where the clinical trial was conducted.

The team found that 24 to 48 hours after the nanoparticles were administered, they had localized in the tumor tissues and released their drug cargo, and the drug had had the intended biological effects of inhibiting two proteins that are involved in the progression of the cancer. Equally important, both the nanoparticles and the drug were absent from healthy tissue adjacent to the tumors.

The nanoparticles are designed to be flexible delivery vehicles. "We can attach different drugs to the nanoparticles, and by changing the chemistry of the bond linking the drug to the nanoparticle, we can alter the release rate of the drug to be faster or slower," says Andrew Clark, a graduate student in Davis's lab and the study's first author.

Davis says his team's findings suggest that a phenomenon known as the enhanced permeability and retention (EPR) effect is at work in humans. In the EPR effect, abnormal blood vessels that are "leakier" than normal blood vessels in healthy tissue allow nanoparticles to preferentially concentrate in tumors. Until now, the existence of the EPR effect has been conclusively proven only in animal models of human cancers.

"Our results don't prove the EPR effect in humans, but they are completely consistent with it," Davis says.

The findings could also help pave the way toward more effective cancer drug cocktails that can be tailored to fight specific cancers and that leave patients with fewer side effects.

"Right now, if a doctor wants to use multiple drugs to treat a cancer, they often can't do it because the cumulative toxic effects of the drugs would not be tolerated by the patient," Davis says. "With targeted nanoparticles, you have far fewer side effects, so it is anticipated that a drug combination can be selected based on the biology and medicine rather than the limitations of the drugs."

These nanoparticles are currently being tested in a number of phase-II clinical trials. (Information about trials of the nanoparticles, denoted CRLX101, is available at www.clinicaltrials.gov).

In addition to Davis and Clark, other coauthors on the study, entitled "CRLX101 nanoparticles localize in human tumors and not in adjacent, nonneoplastic tissue after intravenous dosing," include Devin Wiley (MS '11, PhD '13) and Jonathan Zuckerman (PhD '12); Paul Webster of the Oak Crest Institute of Science; Joseph Chao and James Lin at City of Hope; and Yun Yen of Taipei Medical University, who was at City of Hope and a visitor in the Davis lab at the initiation of the clinical trial. The research was supported by grants from the National Cancer Institute and the National Institutes of Health and by Cerulean Pharma Inc. Davis is a consultant to and holds stock in Cerulean Pharma Inc. 

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Quintessentially Caltech

How best to recognize Caltech's own Ahmed Zewail, the Linus Pauling Professor of Chemistry and professor of physics, and director of the Physical Biology Center for Ultrafast Science and Technology, who has served on Caltech's faculty for 40 years? President Thomas F. Rosenbaum had the answer: what he would later call a "quintessentially Caltech conference."

And so, on Friday, February 26, more than 1,000 people gathered to hear exceptional researchers, including 5 Nobel Laureates, from across disciplines consider our future as part of the full-day "Science and Society" conference that honored the career of Zewail, whom Rosenbaum called "a wizard of scientific innovation."

Read the full story and view the slideshow

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Quintessentially Caltech

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Caltech: A Personal Perspective

Ahmed Zewail, Nobel Laureate
Linus Pauling Professor of Chemistry and Professor of Physics, Caltech

Zewail provided an overview of his journey from a young child in Egypt to Caltech Nobel laureate. On the day he won the 1999 Nobel Prize in Physics, he recalled, Caltech president David Baltimore came to Zewail's house, but he refused to open the door. "We thought he was paparazzi," Zewail admitted.

Credit: Chris Sabanpan

The End of Disease?

Roger Kornberg, Nobel Laureate
Mrs. George A. Winzer Professor in Medicine, Stanford, School of Medicine

As advanced as we think we are, Kornberg said, scientists today understand less than 1 percent of human biology. Attracting more young people to the field of medical research is therefore critical. "Young scientists are the most likely to discover something," he said. "And numbers matter."

Credit: Chris Sabanpan

The Future of Medicine

David Baltimore, Nobel Laureate
Caltech President Emeritus
Robert Andrews Millikan Professor of Biology, Caltech

The human body can survive a maximum of roughly 120 years, according to Baltimore. He predicted a future in which scientists work to push that envelope, using gene editing "to liberate us from the process of aging" and "to perfect the human body, whatever that means."

Credit: Chris Sabanpan

The Future of Quantum Physics

H. Jeff Kimble, Member, National Academy of Sciences
William L. Valentine Professor and Professor of Physics, Caltech

Kimble's lecture about the future of quantum physics included predictions about quantum computing, quantum simulation, and quantum metrology. "Science helps hold us together and appreciate our sameness rather than our differences," he said.

 

Credit: Chris Sabanpan

Time, Einstein, and the Coolest Stuff in the Universe

William Phillips, Nobel Laureate
Physicist, National Institute of Standards and Technology
Distinguished University Professor, University of Maryland

In a hands-on demonstration, Phillips put on a pair of lab goggles and dunked a variety of items—a rose, a racquetball, several inflated balloons—into a vat of liquid nitrogen to help demonstrate his overall point: that we can create super-accurate atomic clocks by cooling down atoms to astoundingly low temperatures.

Credit: Chris Sabanpan

Inequality and World Economics

A. Michael Spence, Nobel Laureate
Philip H. Knight Professor and Dean, Emeritus
Stanford University Graduate School of Business

Spence discussed a number of global economic trends—including the decline in middle-class jobs and the rise of job-eliminating technologies—in a lecture that considered the disparities between rich and poor. "I'm a little worried about what's going on in the global economy right now and I tend to be an optimist," he said.

Credit: Chris Sabanpan

The Future of Space Exploration

Charles Elachi, NASA Outstanding Leadership Medal Recipient
Caltech Vice President
Director, Jet Propulsion Laboratory

Elachi said he believes we will establish a space station on Mars and that humans will begin visiting the planet by 2030. But, he noted, "It's important that we take care of our own planet. It's the only thing we have, at least for now."

 

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How best to recognize Caltech's own Ahmed Zewail, the Linus Pauling Professor of Chemistry and professor of physics, and director of the Physical Biology Center for Ultrafast Science and Technology, who has served on Caltech's faculty for 40 years? President Thomas F. Rosenbaum had the answer: what he would later call a "quintessentially Caltech conference."

And so, on Friday, February 26, more than 1,000 people gathered to hear exceptional researchers, including 5 Nobel Laureates, from across disciplines consider our future as part of the full-day "Science and Society" conference that honored the career of Zewail, whom Rosenbaum called "a wizard of scientific innovation."

The speakers lectured on a broad spectrum of topics, ranging from space travel to global economic inequality to what happens when five inflated balloons are stuffed into a vat of liquid nitrogen. Their talks were moderated by Nathan Gardels, editor in chief of The WorldPost, and Peter Dervan, the Bren Professor of Chemistry, who noted while introducing Zewail that they have been close friends ever since their early days starting as assistant professors together at Caltech.

"What an extraordinary day," Rosenbaum said at the conclusion of the event, held in Beckman Auditorium. "It's unusual to find a series of talks at this incredibly high level of excellence—intellectually deep and pedagogically engaging."

As many of the speakers pointed out, Zewail's list of accomplishments is staggering. He has authored some 600 articles and 16 books and was sole recipient of the 1999 Nobel Prize for his pioneering work in femtochemistry. In the post-Nobel era, he developed a new field dubbed four-dimensional electron microscopy. He has been active in global affairs, serving as the first U.S. Science Envoy to the Middle East and helping establish the Zewail City of Science and Technology in Cairo, which he hopes to turn into "the Caltech of Egypt."

"Ahmed is a very special kind of scientist," said Fiona Harrison, chair of Caltech's Division of Physics, Mathematics and Astronomy, during the conference's introductory remarks. She noted the "incredible breadth of his research" and cited a colleague's observation that "Ahmed is someone who never has average goals."

Jackie Barton, chair of Caltech's Division of Chemistry and Chemical Engineering, praised Caltech for taking a chance on Zewail four decades ago, when he was a young scientist. "He had this vision," she said. "The vision was to watch the dynamics of chemical reactions, to watch reactions happening on a faster and faster time scale, indeed to watch the making and breaking of chemical bonds."

She added: "He has this intuitive sense of the dynamical motions of atoms and molecules, their coherence, or lack thereof, as the case may be. And then he has this extraordinary attention to every detail, so that he's able to meld together theory and experiment and understand that dance, that choreography of atoms and molecules as they carry out a reaction."

To further honor Zewail, Caltech presented him with a rare book of Benjamin Franklin's speeches and scientific research—on lightning rods and the aurora borealis, among other phenomena—that is signed by Rosenbaum and all of Caltech's former presidents. Caltech Provost Ed Stolper noted that it is the only book authored by Franklin that was published during his lifetime.

As Stolper noted in his introductory remarks, the gift is a fitting one for Zewail, who has come to embody the ideal of Caltech, a place "where scientists and engineers are limited only by their imagination." He added Ahmed is one of the few scientists that, like Benjamin Franklin and Linus Pauling, not only excelled in science but has made a broader impact on society through his writings and actions.

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New CCE Leadership Chair Honors the Past and Supports the Future

A new leadership chair in Caltech's Division of Chemistry and Chemical Engineering (CCE) will amplify the Institute's support of its scholars' freedom to pursue the most interesting and challenging lines of inquiry. Established through a $10 million gift from a couple who wish to remain anonymous, the chair will be named in honor of the late Norman Davidson, a longtime Caltech faculty member whose scientific contributions represented in part the beginnings of molecular biology. 

"Through this gift, CCE can maintain its excellence and move forward in new frontiers in chemistry," says Jacqueline K. Barton, Caltech's Arthur and Marian Hanisch Memorial Professor of Chemistry and inaugural holder of the Norman Davidson Leadership Chair.

Read the full story at giving.caltech.edu

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A new leadership chair in the Division of Chemistry and Chemical Engineering (CCE) will amplify the Institute’s support of its scholars’ freedom.

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