Genetic Engineering & Biotechnology News

MAY1 2015

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Genetic Engineering & Biotechnology News | GENengnews.com | MAY 1, 2015 | 15 release of pore-forming toxins by Staphy- lococcus aureus, Vibrio cholera, and Strep- tococcus pyogenes activates the NLRP3 in- fammasome. "This infammasome has received much attention because it also has been linked to pathogenesis of autoinfammatory syn- dromes and a variety of infammatory dis- eases," remarks Dr. Núñez. A number of important questions remain about how infammasomes are involved in both health and disease. According to Dr. Núñez, these questions will be resolved only if scientists secure a more basic understand- ing of the biology and mechanisms of infam- masome activation. "[We need] to know to what extent in- fammasomes are involved in widespread conditions such as diabetes and atheroscle- rosis. We also need to determine how in- fammasomes interact with other signaling pathways to orchestrate innate and adaptive immune responses," explains Dr. Núñez. "As we gain more information about infam- masomes, we may be able to design and de- velop small molecules to block or delay these infammatory diseases." Signalosomes and SMOCs More than 100 years ago, Paul Ehrlich hypothesized that receptors were cellular communicators that were capable of rec- ognizing distinct chemical structures such as toxins. We now know that receptors are sophisticated sensing components involved in multiple signaling pathways, and that they are even more complex than previously imagined. Scientists today are investigating the mo- lecular mechanisms underlying signal trans- duction by immune receptors. One such sci- entist is Hao Wu, Ph.D., professor of biologi- cal chemistry and molecular pharmacology at Harvard Medical School. He notes that the binding of ligands induces conforma- tional changes in receptors, and he adds that such binding is often thought to involve the creation of receptor dimers and trimers that subsequently activate downstream signal- ing cascades. This traditional view, however, may be incomplete. "We discovered a new scenario while we were working on crystallizing receptors," Dr. Wu reports. "During their isolation, we found there were other proteins co-purifying. At frst we thought this was an artifact, but then we realized these were real assemblies." Dr. Wu asserts that his team conducted structural studies that uncovered a vast net- work of higher-order signaling machines, or signalosomes: "We found that these higher- order complexes formed by helical symmetry and that they served to induce the activation of enzymes such as caspases, kinases, and ubiquitin ligases that then lead to cell death, cytokine maturation, and a host of infam- matory responses." Although it was known that receptors assemble into defned oligomers (such as oc- curs during apoptosis), the discovery of large and virtually "infnite" assemblies that have acquired signaling capabilities was novel. "These signaling machines provide unique mechanisms to impart threshold responses and a means for temporal and more spatial control of signaling," Dr. Wu emphasizes. "Further, this opens the door to suggest high- er order assemblies may be an important as- pect of many other biological processes since they promote the formation of very precisely ordered constituents that were initially pres- ent in low concentration of inactive states." According to Dr. Wu, additional studies further expand the new signaling paradigm: "On the basis of structural and cellular stud- ies, we now believe there are supramolecular organizing centers (SMOCs) that are assem- bled on various membrane-bound organelles such as the actin network. These serve to in- crease local concentrations of signaling com- ponents to amplify what would otherwise be weak interactions." Overall, these studies provide a new way of thinking about immune network signaling. Besides being widely used in signal transduc- tion in the immune system, higher-order sig- naling may characterize a general mechanism used by cells for numerous other biological functions. "This is such an elegant mecha- nism, why wouldn't other cells follow it? Cer- tainly, we have only begun to see the tip of the signaling iceberg," concludes Dr. Wu. Cas9 protein Cas9 Nuclease, S. pyogenes, a key protein involved in CRISPR/Cas9 genome editing, is now available from NEB. The release of Cas9 expands the portfolio of products from NEB that support CRISPR workfows, including sgRNA cloning, transcription and HDR template construction. Download our new Genome Editing Brochure at www.neb.com/GenomeEditing Purifed, recombinant protein for genome editing applications NEW ENGLAND BIOLABS ® and NEB ® are registered trademarks of New England Biolabs, Inc. DNA sgRNA PAM (NGG) Nucleotide position (upstream of PAM) 5´ 3´ 5´ 3´ 3´ 5´ Cas9 Nuclease, S. pyogenes Cleavage Cleavage CGCUUGUUUCGGCGUGG 20 15 10 5 1 TGG GTA GCGAACAAAGCCGCACC CGCTTGTTTCGGCGTGG CATACC GUA Schematic representation of Cas9 Nuclease, S. pyogenes sequence recognition and DNA cleavage DRUG DISCOVERY

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