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RESEARCH BRIEFSCrystal Structure Sheds Light on AngiogenesisIn the Oct. 28 Journal of Cell Biology, HMS researchers report the 3-D crystal structure of a protein domain--the thrombospondin type 1 repeat, or TSR--that could lead to the development of future drug therapies to boost or suppress angiogenesis. First author Kemin Tan, HMS research fellow in medicine at the Dana-Farber Cancer Institute, and colleagues elucidated the structure from three TSRs that lie in the extracellular glycoprotein, thrombospondin-1, which has been shown to suppress tumor growth and inhibit angiogenesis.
The structure of the thrombospondin-1 glycoprotein might explain how it inhibits angiogenesis. Recently, HMS researchers have elucidated the crystal structure of a key domain of thrombospondin-1, the type 1 repeat, which interacts with proteins such as transforming growth factor-beta and CD36. The domain has three entwined protein strands surrounding an eight-layered core, which features the novel stacking of tryptophans (W) and arginines (R), capped by cysteines (C). (Image courtesy of Kemin Tan) The unique folding motif of the TSR, a domain involved in cell proliferation, migration, and death, sheds light on thrombospondin-1 and the approximately 50 other human proteins that also contain copies of this domain. Co-senior author Jack Lawler, HMS associate professor of pathology at Beth Israel Deaconess Medical Center, who has investigated thrombospondin-1 for more than 20 years, explained, "We know that genetic mutations of tumor cells are associated with a decreased expression of thrombospondin-1. Knowing the structure of TSRs means we can further examine the protein on a molecular level and see how it activates proteins, such as transforming growth factor-beta, and induces apoptosis after binding to CD36, an endothelial cell membrane protein." The study, based on high-resolution X-ray crystallography, revealed that the TSR is composed of three intertwined protein strands stabilized by hydrophobic amino acids. When the researchers probed further, they found a unique core structure, which they later dubbed CWR for the single-letter abbreviations of its predominant amino acids, cysteine, tryptophan, and arginine. The side chains of the amino acids interdigitate to form an organized, eight-layered structure with alternately stacked tryptophans and arginines sandwiched between cysteines. "To the best of our knowledge, the major structural feature of tryptophan and arginine layers offers a new structural domain in the protein world," said Jia-huai Wang, HMS associate professor of pediatrics (biological chemistry and molecular pharmacology) at Dana-Farber, in whose laboratory the structure was uncovered. The researchers also found a positively charged groove on the domain, which they propose is a docking site for other molecules. The team is currently attempting to crystallize these complexes and determine the crystal structure of other domains on thrombospondin-1. --Trang Au
Small Molecules Confound Lipid-Transferring Ability Of 'Good' CholesterolResearchers at the Center for Blood Research and MIT have found new tools that may help unmask the molecular activities of the "good" high-density lipoprotein cholesterol at its receptor, scavenger receptor, type I (SR-BI). In animal and cell studies, HDL appears to take cholesterol out of body tissues, including the atherosclerotic plaques in artery walls, and deliver it to liver cells, which can ship it out of the body via the intestines in the form of bile. The receptor, which also binds other molecules, binds cholesterol-laden HDL particles, extracts the cholesterol esters from the fatty core, and releases the fat-depleted particles into the extracellular space. Researchers in the labs of Tom Kirchhausen, HMS professor of cell biology at the Center for Blood Research, and Monty Krieger, professor of biology at MIT (who discovered SR-BI and its role as an HDL receptor), identified five small molecules they call BLTs, named for their ability to block lipid transport. In the presence of BLTs, HDL can bind to the receptor but cannot exchange lipids with cells, the team reports in the Nov. 19 online edition (and Nov. 26 print edition) of Proceedings of the National Academy of Sciences. In the study, first author Thomas Nieland, a graduate student in Kirchhausen's group, who has been working in both laboratories, and his coworkers screened a library of 16,320 small molecules at the Institute for Chemistry and Cell Biology at HMS. They found five especially potent chemicals that blocked both the transfer of lipid from HDL to cells and the transfer of lipid from cells to HDL. Kirchhausen and Krieger believe the results offer proof of principle that receptor activators and other inhibitors might be found through similar techniques. More immediately, the chemicals offer precise tools to reveal how HDL works through SR-BI to do its physiological good works, including influencing atherosclerosis, ovary function, and red blood cell development. --Carol Cruzan Morton
HMS Lends Hand to Landmark Mouse Genome StudyResearchers at HMS contributed to last week's landmark publication of the mouse genome and related papers. Scientists expect the genome will provide information and tools to greatly accelerate research into human biology and diseases. The bulk of the high-quality draft sequence made public in the Dec. 5 Nature and deposited at several public websites was produced by the relatively fast and cheap technique of whole-genome shotgun sequencing at MIT's Whitehead Institute, Washington University in St. Louis, and the Wellcome Trust Sanger Institute in the U.K. The draft sequence was verified and supplemented by a number of independent methods, including sequences from the Harvard Medical School-Partners Center for Genetics and Genomics and other smaller groups from the nine NIH-funded members of the Mouse Genome Sequencing Consortium. The Harvard Center started sequencing from the other direction, selecting mapped bacterial artificial chromosome (BAC) clones, sequencing them to a high degree of accuracy, and adding their results to the larger effort. The center moved to Harvard last year from the Albert Einstein College of Medicine, where it had also contributed to the public human genome project by mapping human chromosome 12. "We set out to sequence biologically interesting regions of the genome for ourselves and for a number of researchers around the country," said center scientific director Raju Kucherlapati, the Paul C. Cabot professor of genetics and professor of medicine at HMS and Brigham and Women's Hospital. Kucherlapati and Kate Montgomery, HMS instructor in medicine at BWH, are listed as co-authors of the mouse genome paper and represent the efforts of many other people at the center, Montgomery said. In Kucherlapati's lab, researchers have already used the results to generate mouse models with mutations in genes involved or implicated in the onset and progression of human colorectal cancer, velo-cardio-facial syndrome, and other diseases. Meanwhile, another international consortium has published the first complete draft of the working parts of a mammalian genome in the same issue of Nature. Genes are often defined as protein-coding regions, but the FANTOM consortium (mostly based at RIKEN Genomic Sciences Center in Japan) leveraged a technique to synthesize full-length cDNA clones and found that almost one third of the mouse "transcriptome" is never converted into proteins. They may function in their RNA form to switch other genes on and off at specific times and places. Stefano Gustincich, HMS instructor in neurobiology, was a collaborator and co-author. As part of the FANTOM consortium, Gustincich contributed retinal tissues and annotated his share of the project's sequenced cDNA. In collaboration with Elio Raviola, the Bullard professor of neurobiology, Gustincich has already used a resulting tool, full-length clones in a cDNA microarray, to identify more than 700 genes expressed in a single dopamine neuron in the mouse retina in an effort to understand how high levels of dopamine regulate light adaptation during the light of day and drop off in the dark of night. --Carol Cruzan Morton |
