Dr. Dohlman is currently an editorial board member of JBC with an area of expertise in signal transduction. He will serve as associate editor for 5 years, starting July 1, 2013. Our congratulations to Dr. Dohlman!

Wednesday, April 10, 2013

CHAPEL HILL, N.C. – Researchers at the University of North Carolina at Chapel Hill School of Medicine have “rationally rewired” some of the cell’s smallest components to create proteins that can be switched on or off by command. These “protein switches” can be used to interrogate the inner workings of each cell, helping scientists uncover the molecular mechanisms of human health and disease.

In the first application of this approach, the UNC researchers showed how a protein called Src kinase influences the way cells extend and move, a previously unknown role that is consistent with the protein’s ties to tumor progression and metastasis.

Nikolay Dokholyan, PhD

“This rationally designed control of protein conformations represents a breakthrough in computational protein design,” said senior study author Nikolay Dokholyan, PhD, a professor of biochemistry and biophysics. “We now have a new tool for delineating the activities of various proteins in living cells in a way that was never before possible.”

The research was published online ahead of print in the Proceedings of the National Academy of Sciences. In the study, Dokholyan created a “switch” that would make a protein wobbly and unable to do its job unless it was flipped “on” by a drug called rapamycin, which would stabilize the protein and let it perform its function.

The approach is a simpler and more reliable version of a protein engineering system pioneered three years ago by Dokholyan and Klaus Hahn, professor of pharmacology at UNC, called rapamycin regulated or RapR.  In the old approach, the switching mechanism depended on two proteins and the drug. The first protein – the one the researchers wanted to study – was given the RapR modification and put in cells in tissue culture. The second protein was placed in the cells as well, but simply floated around until the addition of drug caused it to latch on to the modification in the first protein and turn it on. The problem with the approach was that some cells would have a lot of the first protein and less of the second, or vice versa.

“It became the Achilles heel of the technique, because there was variability in the results due to the different ratios between the proteins,” said Hahn. “What Dokholyan was able to do, which was extremely challenging from a protein engineering standpoint, was to combine the two parts into one.” Dokholyan and Hahn are members of the UNC Lineberger Comprehensive Cancer Center.

Dokholyan and his colleagues took the two proteins and broke them apart into their individual components, structures called alpha helices and beta sheets. They then rewired them together to make a whole new protein where the parts could interact with each other. When researchers compared this system, called uniRapR, with the previous approach, they found the new one gave cleaner, more reliable and more consistent results.

Onur Daglyan, co-first author on the paper along with David Shirvanyants.

Working both in cultured human cells and in the model organism zebrafish, the researchers showed that turning on Src causes the cell to extend its edges as part of cell movement. Now that they have dissected the role of one protein, the researchers plan to look at a variety of other kinases to understand their roles in the development, progression, and spread of cancer.

The research was funded in by the National Institutes of Health, the National Institute of Environmental Health Sciences, and the National Cancer Institute. Study co-authors from UNC were Onur Dagliyan; David Shirvanyants, PhD; Andrei V. Karginov, PhD; Feng Deng, PhD; Lanette Fee; and Srinivas N. Chandrasekaran. Co-authors from the University of Wisconsin, Madison, were Christina M. Freisinger, Gromoslaw A. Smolen, and Anna Huttenlocher.

LA JOLLA, CA – March 21, 2012 ­– A team including scientists from The Scripps Research Institute (TSRI), the University of North Carolina at Chapel Hill and the Chinese Academy of Sciences has determined and analyzed the high-resolution atomic structures of two kinds of human serotonin receptor. The new findings help explain why some drugs that interact with these receptors have had unexpectedly complex and sometimes harmful effects.

“Understanding the structure-function of these receptors allows us to discover new biology of serotonin signaling and also gives us better ideas about what biological questions to probe in a more intelligent manner,” said TSRI Professor Raymond Stevens, who was a senior investigator for the new research. The studies were published in two papers on March 21, 2013 in Science Express, the advance online version of the journal Science.

Stevens’s laboratory at TSRI has pioneered the development of techniques for determining the 3D atomic structures of cellular receptors—particularly the large receptor class known as G protein-coupled receptors (GPCRs). GPCRs sit in the cell membrane and sense various molecules outside cells. When certain molecules bind to them, the receptor's respond in a way to transmit a signal inside the cell. Approximately one third of all drugs target GPCRs.

In the past several years, using X-ray crystallography, the Stevens laboratory has determined the high-resolution structures of ten of the most important GPCRs for human health— including the β2 adrenergic receptor, the A2a adenosine receptor (the target of caffeine), HIV related CXCR4 receptor, the pain-mediating nociceptin receptor, S1P1 receptor important for inflammatory diseases,  and the D3 dopamine receptor which is involved in mood, motivation and addiction.

Serotonin receptors are no less important. “Nearly all psychiatric drugs affect serotonin receptors to some extent, and these receptors also mediate a host of effects outside the brain, for example on blood coagulation, smooth muscle contraction and heart valve growth,” said Bryan Roth, a collaborator on both studies who is professor of pharmacology at the University of North Carolina (UNC).

Untangling Two Serotonin Receptors

Roth’s laboratory teamed up with Stevens’s as part of the National Institute of General Medical Sciences (NIGMS) Protein Structure Initiative. For this project the two labs also worked with the laboratories of Professors Eric Xu and Hualiang Jiang, at the Shanghai Institute of Materia Medica, part of the Chinese Academy of Sciences. “By collaborating with the Chinese teams we were able to complete a much more through study and get the most out of our fundamental structural results,” said Stevens.

In the first of the new studies, co-lead author Chong Wang, a graduate student in the Stevens laboratory, and his colleagues determined the structure of the serotonin receptor subtype 5-HT_1B, the principal target of several drug classes. (5-HT, or 5-hydroxytryptamine, is a technical term for serotonin.) The team produced the 5-HT_1B receptor while it was bound by either ergotamine or dihydroergotamine—two old-line anti-migraine drugs that work in part by activating 5-HT_1B receptors.

With the help of the special fusion protein, nicknamed BRIL (apocytochrome b562RIL), Wang and colleagues were able to stabilize these structures and coax them to line up in a regular ordering known as a crystal. X-ray crystallography revealed, at high resolution, an atomic structure of 5-HT_1B with a main binding pocket and a separate, extended binding pocket.

Harmful Off-Target Effects

In the second study, TSRI graduate student and lead author Daniel Wacker and colleagues used similar techniques to determine the structure of the 5-HT_2B receptor bound to ergotamine.

The 5-HT_2B receptor was chiefly of interest because drug developers want to avoid activating it. “Drugs that are meant to target other serotonin receptors in the brain can have harmful off-target effects on 5-HT_2B receptors, which are found abundantly on heart valves, for example,” said Roth. The weight-loss drug fenfluramine and closely related dexfenfluramine were withdrawn from the US market in 1997 after being linked to heart valve disease. Roth’s laboratory later showed that this side effect was mediated by heart valve 5-HT_2B receptors.

Analyses of the 5-HT_1B and 5-HT_2B receptor structures revealed a subtle difference between them. “Although their main binding pockets look very similar, their extended binding pockets are not as similar—the one for 5-HT_2B is narrower and in a slightly different position,” said Wang.

With the two receptor structures in hand, the Xu and Jiang team simulated the bindings of various drugs. They showed, for example, that anti-migraine drugs called triptans should bind well to 5-HT_1B receptors but poorly to 5-HT_2B receptor structures, in which the extended binding pocket is less accessible. Similarly, the team’s calculations confirmed that fenfluramine’s active metabolite should bind very tightly to the 5-HT_2B receptor.

Delving Deeper

In the second study, the researchers used the 5-HT_2B and 5-HT_1B structural data to better understand a recently discovered GPCR signaling pathway.

When a neurotransmitter such as serotonin binds to its GPCR receptor and triggers the primary, G protein-mediated activation signal, it also usually triggers another signal, often mediated by a protein called β-arrestin. This second signaling cascade may simply have the effect of “arresting” or inhibiting the primary, G protein-mediated signaling. But it can also have other effects on the cell, and although most molecules bind to their target GPCRs in a way that activates these primary and secondary signals equally, others preferentially activate one or the other. “Such functional selectivity, as we call it, adds another layer of complexity to drug effects on GPCRs,” said Roth, a co-senior author of the study.

Roth’s laboratory produced several 5-HT receptor subtypes in test cells, and compared the strength of G-protein and β-arrestin signaling when these receptors were bound by ergotamine or various other drugs, including the ergotamine-derived hallucinogen LSD (lysergic acid diethylamide). Most of the tested drugs showed no bias. However, ergotamine, LSD and some of their relatives turned out to be clearly biased in favor of β-arrestin signaling at the 5-HT_2B receptor. Comparison of the ergotamine-bound 5-HT_2B structure with the ergotamine-bound 5-HT_1B structure revealed the likely reason. “We could see that when ergotamine is bound to the 5-HT_2B receptor it stabilizes the receptor structure in a conformation that interferes with G protein signaling,” said Wacker.

The findings allow scientists to start probing this arrestin-mediated signaling pathway and its downstream effects in a more targeted manner. “These structural data are teaching us to ask better questions about receptor biology,” said Stevens.

In addition to Chong, the two other first authors of the first study, “Structural Basis for Molecular Recognition at Serotonin Receptors,” were Yi Jiang, a researcher in the Stevens laboratory who was visiting from the Xu laboratory in Shanghai, and Jinming Ma, a researcher in the Stevens laboratory who was visiting from the Van Andel Research Institute in Michigan, where Xu runs a laboratory. Other contributors to this study were Huixian Wu, Daniel Wacker, Vsevolod Katritch, Gye Won Han, Wei Liu and Vadim Cherezov of TSRI; Xi-Ping Huang, Eyal Vardy and John D. McCorvy of Roth’s laboratory at UNC; Xiang Gao, Edward X. Zhou, Karsten Melcher and Chenghai Zhang of the Van Andel Research Institute; Fang Bai of the Dalian University of Technology in China; and Huaiyu Yang, Linlin Yang of Xu and Jiang laboratories in Shanghai.

Contributors to the second study, “Structural Features for Functional Selectivity at Serotonin Receptors,” included Chong Wang, Vsevolod Katritch, Gye Won Han, Meihua Chu, Fai Yiu Siu, Wei Liu, Yi Jiang and Vadim Cherezov of TSRI; Xi-Ping Huang, Eyal Vardy and John D. McCorvy of the Roth laboratory at UNC; and Eric Xu.

Support for these studies was provided by the National Institute of General Medical Sciences (PSI:Biology grant U54 GM094618), the National Institutes of Health Common Fund in Structural Biology (P50 GM073197), the Jay and Betty Van Andel Foundation, the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK071662), Chinese Ministry of Science and Technology (grants 2012ZX09301001-005 and 2012CB910403); Amway (China); the National Institute of Mental Health (NIMH) (R01 MH61887, U19 MH82441), the National Institute on Drug Abuse (R01 DA27170) and the NIMH Psychoactive Drug Screening Program.

Links to the 2 papers on the Science Magazine Express website:

"Structural Features for Functional Selectivity at Serotonin Receptors," Daniel Wacker, et al. Science, published online March 21, 2013.

"Structural Basis for Molecular Recognition at Serotonin Receptors," Chong Wang, et al. Science, published online March 21, 2013.

Links to other articles highlighting the papers:

Nature Journal highlights the two Science papers describing serotonin structure and its effects

Chemical and Engineering News (CEN) highlights the two articles

To date, there is no cure or treatment for Canavan disorder.

In a collaborative research effort with Dr. P Leona investigative team at UNDMJ, the Samulski lab designed an AAV vector that efficiently transduces neuronal cells carrying the ASPA transgene under CMV promoter. After demonstrating safety and efficacy in non-human primates the Samulski lab produced FDA approved GMP vector in the UNC Vector core facility for Phase I clinical trial which was used in the first neuronal gene therapy trial in the US. The outcome of these studies were recently published in Science Translation were the investigators found that the gene transfer approach was safe, decreased N-acetyl-asparate, seizure frequency and provided clinical stabilization. Clinical stabilization was greatest in the youngest patients supporting that early detection and treatment with gene therapy-mediated enzyme replacement in neonatal period may offer the best opportunity for reduction of symptoms and long-term stability in patients with Canavan disease.

Full citation for the paper:

Paola Leone, David Shera, Scott W.J. McPhee, Jeremy S. Francis, Edwin H. Kolodny, Larissa T. Bilaniuk, Dah-Jyuu Wang, Mitra Assadi, Olga Goldfarb, H. Warren Goldman, Andrew Freese, Deborah Young, Matthew J. During, R. Jude Samulski and Christopher G. Janson.  Long-Term Follow-Up After Gene Therapy for Canavan Disease.  Sci Transl Med,  Vol. 4, Issue 165, p. 165ra163, 2012. DOI: 10.1126/scitranslmed.3003454.

Read article on North Carolina Biotechnology website: "UNC's Samulski Parlays NCBiotech Support Into Cutting-Edge Gene Therapies"

Read article on the DNA Science Blog on the PLOS website: "Gene Therapy for Canavan Disease: Max’s Story"

Read article for Genomics on SciCasts website: "Gene Therapy Cocktail Shows Promise in Long-term Clinical Trial for Canavan Disease"

The Youtube video below was released by Science Translational Medicine Press and is a trailer adapted from a documentary to be released sometime in 2013.

"Dark matter made visible before the final cut

Research findings from the University of North Carolina School of Medicine are shining a light on an important regulatory role performed by the so-called dark matter, or "junk DNA," within each of our genes. The new study reveals snippets of information contained in dark matter that can alter the way a gene is assembled."

The full citation for the publication is: Nature Structural & Molecular Biology 19: 1044-1052, 2012, doi: 10.1038/nsmb.2377.

Yang Wang, Research Assistant Professor in the Wang lab, is first author on paper.  Along with Zefeng Wang, co-authors are Meng Ma, from UNC and Xinshu Xiao at UCLA.

Read more about the original paper

Read a UNC School of Medicine News article highlighting the paper

Watch the video on the Nobel Website: Unlocking the Secrets of Our Cells: The Nobel Prize
Dr. Roth's interview begins at 25:05 into the 30 minute video on the ground breaking work of 2012 Chemistry Laureates Robert J. Lefkowitz and Brian K. Kobilka and 2012 Medicine Laureates Sir John B. Gurdon and Shinya Yamanaka and how it has influenced leading scientists and improved the lives of patients.

Photo below: Dr. Bryan Roth being interviewed in Roth Lab by Richard Wyllie of Blakeway Productions for Bloomberg TV. Click for larger image.

Photo below: Hui Wang with live cell imaging demo in one of the Hahn Lab microscope rooms. Click for larger image.

Half the science writers heard a presentation by Klaus Hahn on the imaging being done in the lab while the other half saw a demonstration of live cell imaging being manipulated by light by Hui Wang in the Hahn Lab microscope room.  Then the groups switched places. The visit was arranged by UNC.

Photo below: Klaus Hahn presents Hahn lab live cell imaging research to science writers. Click for larger image.

Photo below: Live cell imaging showing photoactivation of cellular processes. Click for larger image.

Read the paper by Arthur, et al., in  Science, 338(6103):120-3, 2012.

An article about the paper is published in the Carolina Alumni Review and in the Endeavors magazine online Nov. 29, 2012: Gut Busters.

Photo below from the Endeavors article is of Jobin lab member, Ernesto Perez-Chanona, a Pharmacology graduate student.  Photo by Donn Young.

The Wang Lab had a paper published in Nature Structural & Molecular Biology September 16, 2012 titled, "Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules." Yang Wang, Research Assistant Professor in the Wang lab, is first author on paper.  Along with Zefeng Wang, co-authors are Meng Ma, from UNC and Xinshu Xiao at UCLA. Congratulations to all!

Nature Structural & Molecular Biology (2012) doi:10.1038/nsmb.2377

From the papers abstract:

Most human genes produce multiple splicing isoforms with distinct functions. To systematically understand splicing regulation, we conducted an unbiased screen and identified >100 intronic splicing enhancers (ISEs), clustered by sequence similarity. All ISEs functioned in multiple cell types and in heterologous introns, and patterns of distribution and conservation across pre-mRNA regions were similar to those of exonic splicing silencers. Consistently, all ISEs inhibited use of splice sites from exons. Putative trans-factors of each ISE group were identified and validated. Five distinct groups were recognized by hnRNP H and hnRNP F, whose C-terminal domains were sufficient to render context-dependent activities of ISEs. The sixth group was controlled by factors that either activate or suppress splicing. We provide a comprehensive picture of general ISE activities and suggest new models of how single elements can function oppositely, depending on locations and binding factors.

Figure below: click for enlargement.

The research project which the award will support is described below:

"Precise control of cell cycle progression involves regulation of a large number of genes in a periodic manner. Emerging evidence suggests that cell cycle is controlled by pre-mRNA alternative splicing, however the scope and the mechanisms of splicing regulation in cell cycle remain unclear.  We have recently found that a protein kinase, Clk1, play a critical role in controlling splicing during cell cycle through phosphorylating a main class of splicing factors (i.e. SR proteins).  Clk1 is a cell cycle regulated protein whose level peaks during mitosis. In addition, we found the inhibition of Clk1 changed splicing in hundreds of genes that are essential for cell division and DNA damage response. These observations led to the hypothesis that the temporal control of splicing plays a key role in regulating cell cycle and that Clk1 mediated SR protein phosphorylation is essential for critical alternative splicing events in normal cell cycle progression. Therefore we will determined the SR protein targets of Clk1 that control cell cycle related splicing events, and further define the temporal control of alternative splicing during cell cycle in a genomic scale."

The aim of the Jefferson Pilot Fellowship program is to retain promising junior faculty at the University of North Carolina at Chapel Hill School of Medicine. This is accomplished in two ways. First, the recipient of the award, by being named a Jefferson-Pilot Fellow in Academic Medicine, is given public recognition of his/her prior achievements and the School's confidence in his/her future; second, the monetary award allows him/her a degree of freedom to explore new ideas, new ways of teaching students, treating patients, or investigating biological problems that may not be available from other granting agencies.

Our congratulations to Dr. Wang!

Pilar's laboratory at UNC researched mechanisms of tumorigenesis and tumor progression, and the application of genome-wide techniques to develop anti-cancer therapies. Her research focused on transcriptional regulation of gene expression during stem cell self-renewal and differentiation and during tumorigenesis.

Sabine Stolzenberg, a postdoc in Pilar's lab, will be joining her as part of her new lab.  We wish them both the best in all their adventures and research down under!

Here's a slideshow with pictures from her goodbye party.

Photos below: (left) Sabine and Pilar (right) Gary and Pilar.  Click for enlargement.

Our congratulations to Dr. Tim Elston, John Houser, the first author on the paper, and the other co-authors: Eintou Ford, Michael Nagiec, and Beverly Errede, all from UNC.

Anuj Kumar and Cole Johnson of the F1000 explain the paper's importance thusly:

"In this interesting paper, the authors investigate Ste12p-mediated transcriptional regulation of the yeast pheromone signaling network (mating response). Specifically, the authors utilized integrated computational and experimental approaches to generate an ordinary differential equation (ODE)-based mathematical model that not only accurately mimics the observed in vivo pheromone response of yeast but can also predict the response of this network to genetic perturbations, offering a predictive understanding of the yeast mating response."

"Living organisms display a remarkable ability to quickly and appropriately respond to changes in their extracellular environment. Their response is often mediated through tightly coordinated gene expression using multiple control mechanisms integrated into a complex gene regulatory network. Despite continual research into the design principles of complex signaling networks, our ability to reconstruct the multifaceted gene regulatory networks observed in nature is in its infancy. Here, the authors further our predictive understanding of genetic regulatory networks by combining computational and experimental methodologies to study transcriptional regulation mediated by Ste12 in budding yeast. Specifically, Houser et al. developed a mathematical model that can accurately describe multiple, unique characteristics of the Ste12-mediated pheromone response."

To read the full article explaining the paper's importance go to: http://f1000.com/717948916

About the F1000: "Faculty of 1000 (F1000) identifies and evaluates the most important articles in biology and medical research publications. Articles are selected by a peer-nominated global 'Faculty' of the world's leading scientists and clinicians who then rate them and explain their importance." (from the F1000 Research website: http://f1000.com/about/whatis)

You can find out more about the F1000 at http://f1000.com/tour

David has been with the department since 1999 when he joined as an Assistant Professor after a four-year stint with AMGEN Inc. Now a full Professor, he has contributed greatly to the research of the department, most notably in the area of heterotrimeric G-protein signaling and RGS protein structure and function.  He has lectured for the BBSP pharmacology graduate school curriculum as well as the medical school pharmacology curriculum. Highly regarded for his teaching, he won the Phillip and Ruth Hettleman Prize in 2006 from UNC. In that same year, he became the Thomas J. Dark Basic Research Director of the UNC MD/PhD Program, and in 2009, the Director of Chemical Biology for the Pharmacology department.

Our congratulations to David.  We wish him well in his new leadership position. We are better off for his having been here.

Below are some pictures from his goodbye party.  Click for enlargements or just peruse the slideshow - the link is below the pictures.

Slideshow of pictures from the party

A type of tumor in 20% of breast cancer patients becomes resistant to drugs that are normally effective treatments.

The protein activation test allows you to see how the cancer cells evade the drug.  Once the protein pathway of evasion is known, doctors can try new treatment strategies for patients with drug resistant tumors. The new strategies may try to block the cancer cells' ability to evade the medicine or they may involve  trying a new medicine that the patient may respond to better.

UNC researchers have received a $900,000 grant from the Susan B. Komen For The Cure ® for clinical trials with the protein test.

Watch the news video on the WRAL website.

Read more about the award, clinical applications for the protein kinase activation in  response to inhibitory drugs in HER2-positive breast cancer in UNC School of Medicine News.

View Announcement.

(From the Fulbright Program website):

"The Fulbright Program is the flagship international educational exchange program sponsored by the U.S. government and is designed to “increase mutual understanding between the people of the United States and the people of other countries.” With this goal as a starting point, the Fulbright Program has provided almost 310,000 participants—chosen for their academic merit and leadership potential — with the opportunity to study, teach and conduct research, exchange ideas and contribute to finding solutions to shared international concerns."

"The Fulbright Program awards approximately 8,000 grants annually. Roughly 1,600 U.S. students, 4,000 foreign students, 1,200 U.S. scholars, and 900 visiting scholars receive awards, in addition to several hundred teachers and professionals. Approximately 310,000 "Fulbrighters" have participated in the Program since its inception in 1946."

"Currently, the Fulbright Program operates in over 155 countries worldwide"