Nature Biotechnology

Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo

Nature Biotechnology 28, 839 (2010). doi:10.1038/nbt.1663

Authors: Nathalia Holt, Jianbin Wang, Kenneth Kim, Geoffrey Friedman, Xingchao Wang, Vanessa Taupin, Gay M Crooks, Donald B Kohn, Philip D Gregory, Michael C Holmes & Paula M Cannon

An allosteric inhibitor of substrate recognition by the SCFCdc4 ubiquitin ligase

Nature Biotechnology 28, 733 (2010). doi:10.1038/nbt.1646

Authors: Stephen Orlicky, Xiaojing Tang, Victor Neduva, Nadine Elowe, Eric D Brown, Frank Sicheri & Mike Tyers

The specificity of SCF ubiquitin ligase–mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCFCdc4. A co-crystal structure reveals that SCF-I2 inserts itself between the β-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 Å away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase

Nature Biotechnology 28, 738 (2010). doi:10.1038/nbt.1645

Authors: Mariam Aghajan, Nao Jonai, Karin Flick, Fei Fu, Manlin Luo, Xiaolu Cai, Ikram Ouni, Nathan Pierce, Xiaobo Tang, Brett Lomenick, Robert Damoiseaux, Rui Hao, Pierre M del Moral, Rati Verma, Ying Li, Cheng Li, Kendall N Houk, Michael E Jung, Ning Zheng, Lan Huang, Raymond J Deshaies, Peter Kaiser & Jing Huang

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)Met30 ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCFMet30in vivo and in vitro, but not the closely related SCFCdc4. Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

Engineered allosteric activation of kinases in living cells

Nature Biotechnology 28, 743 (2010). doi:10.1038/nbt.1639

Authors: Andrei V Karginov, Feng Ding, Pradeep Kota, Nikolay V Dokholyan & Klaus M Hahn

Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions (Fig. 1a). Catalytic activity is restored by addition of rapamycin or non-immunosuppresive rapamycin analogs. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.

At ground level

Nature Biotechnology 28, 775 (2010). doi:10.1038/bioe.2010.6

Author: Julian Bertschinger

The hardest—and perhaps loneliest—period of being an entrepreneur might be just after your company is founded.

Pairwise agonist scanning predicts cellular signaling responses to combinatorial stimuli

Nature Biotechnology 28, 727 (2010). doi:10.1038/nbt.1642

Authors: Manash S Chatterjee, Jeremy E Purvis, Lawrence F Brass & Scott L Diamond

Prediction of cellular response to multiple stimuli is central to evaluating patient-specific clinical status and to basic understanding of cell biology. Cross-talk between signaling pathways cannot be predicted by studying them in isolation and the combinatorial complexity of multiple agonists acting together prohibits an exhaustive exploration of the complete experimental space. Here we describe pairwise agonist scanning (PAS), a strategy that trains a neural network model based on measurements of cellular responses to individual and all pairwise combinations of input signals. We apply PAS to predict calcium signaling responses of human platelets in EDTA-treated plasma to six different agonists (ADP, convulxin, U46619, SFLLRN, AYPGKF and PGE2) at three concentrations (0.1, 1 and 10 × EC50). The model predicted responses to sequentially added agonists, to ternary combinations of agonists and to 45 different combinations of four to six agonists (R = 0.88). Furthermore, we use PAS to distinguish between the phenotypic responses of platelets from ten donors. Training neural networks with pairs of stimuli across the dose-response regime represents an efficient approach for predicting complex signal integration in a patient-specific disease milieu.

A mouse knockout library for secreted and transmembrane proteins

Nature Biotechnology 28, 749 (2010). doi:10.1038/nbt.1644

Authors: Tracy Tang, Li Li, Jerry Tang, Yun Li, Wei Yu Lin, Flavius Martin, Deanna Grant, Mark Solloway, Leon Parker, Weilan Ye, William Forrest, Nico Ghilardi, Tamas Oravecz, Kenneth A Platt, Dennis S Rice, Gwenn M Hansen, Alejandro Abuin, Derek E Eberhart, Paul Godowski, Kathleen H Holt, Andrew Peterson, Brian P Zambrowicz & Frederic J de Sauvage

Live attenuated influenza virus vaccines by computer-aided rational design

Nature Biotechnology 28, 723 (2010). doi:10.1038/nbt.1636

Authors: Steffen Mueller, J Robert Coleman, Dimitris Papamichail, Charles B Ward, Anjaruwee Nimnual, Bruce Futcher, Steven Skiena & Eckard Wimmer

Despite existing vaccines and enormous efforts in biomedical research, influenza annually claims 250,000–500,000 lives worldwide, motivating the search for new, more effective vaccines that can be rapidly designed and easily produced. We applied the previously described synthetic attenuated virus engineering (SAVE) approach to influenza virus strain A/PR/8/34 to rationally design live attenuated influenza virus vaccine candidates through genome-scale changes in codon-pair bias. As attenuation is based on many hundreds of nucleotide changes across the viral genome, reversion of the attenuated variant to a virulent form is unlikely. Immunization of mice by a single intranasal exposure to codon pair–deoptimized virus conferred protection against subsequent challenge with wild-type (WT) influenza virus. The method can be applied rapidly to any emerging influenza virus in its entirety, an advantage that is especially relevant when dealing with seasonal epidemics and pandemic threats, such as H5N1- or 2009-H1N1 influenza.

An empiric victory

Nature Biotechnology 28, 529 (2010). doi:10.1038/nbt0610-529

Provenge already looks like the product of a bygone era.

Landmark approval for Dendreon's cancer vaccine

Nature Biotechnology 28, 531 (2010). doi:10.1038/nbt0610-531

Author: Laura DeFrancesco

Firms chase diabetic inflammation with anti-IL-1β antibodies

Nature Biotechnology 28, 533 (2010). doi:10.1038/nbt0610-533

Author: Cormac Sheridan

African GM safety drill

Nature Biotechnology 28, 534 (2010). doi:10.1038/nbt0610-534

Author: Lucas Laursen

Burgeoning stem cell product market lures major suppliers

Nature Biotechnology 28, 535 (2010). doi:10.1038/nbt0610-535

Author: Sarah Webb

GMP cell lines to order

Nature Biotechnology 28, 536 (2010). doi:10.1038/nbt0610-536a

Author: Susan Aldridge

Open-access fermenter

Nature Biotechnology 28, 536 (2010). doi:10.1038/nbt0610-536b

Author: Asher Mullard

Glyphosate resistance threatens Roundup hegemony

Nature Biotechnology 28, 537 (2010). doi:10.1038/nbt0610-537

Author: Emily Waltz

SBIR grants wax

Nature Biotechnology 28, 538 (2010). doi:10.1038/nbt0610-538a

Author: Emma Dorey

Relief over stem cell lines

Nature Biotechnology 28, 538 (2010). doi:10.1038/nbt0610-538b

Author: Jeffrey L Fox

Obama appoints bioethics panel to offer practical advice

Nature Biotechnology 28, 539 (2010). doi:10.1038/nbt0610-539a

Author: Jeffrey L Fox

GSK's RNA splash

Nature Biotechnology 28, 539 (2010). doi:10.1038/nbt0610-539b

Author: Janelle Weaver