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Research Highlights

Shedding light on protein networks

Barrios-Rodiles et al. have added luciferase to the list of markers used in identifying protein-protein interactions in mammalian cells. In doing so, they have created a high-throughput approach that allows the rapid construction of interaction networks. Their strategy involves fusing proteins of interest to Renilla luciferase and then coexpressing the fusions with potential protein interactors bearing 3× Flag epitope tags. Once immunoprecipitated with anti-Flag antibody, positive protein interactions are determined via Renilla luciferase enzymatic assays. The authors applied their system to the transforming growth factor (TGF)-β family of cytokines in which protein-protein interactions are regulated through post-translational modifications. Libraries of 3× Flag-tagged cDNAs were coexpressed in mammalian cells with core members of the TGFβ pathway labeled with luciferase. Using a robotics platform, the authors performed 12,000 experiments, which were analyzed with the help of a clustering algorithm to map protein network profiles. This resulted in the identification of known and novel protein-protein interactions with TGFβ receptors. Novel interactions included those between TGFβ type I receptor and both PAK1 (a member of the p21-activated kinase network) and Occludin (a structural component of tight junctions). Moreover, the authors showed that Occludin modulates the localization of TGFβ type I receptor. This study provides insight into the role that TGFβ receptors play in tight junction dissolution when epithelial cells change into mesenchymal cells during development or tumorigenesis. (Science 307, 1603–1609, 2005) NC

Rules that get under your skin

Chemical permeation enhancers (CPEs) are used to help medicines and cosmetics cross the skin barrier—the most efficient CPEs being typically also the most irritant. Mitragotri and colleagues have now devised a way to predict compound efficiency and irritability to skin, thereby providing a means for designing safer and more potent CPEs. First, through a systematic analysis of existing CPEs by Fourier transform infrared spectroscopy, they show that CPEs work either by extraction or fluidization of lipid bilayers via two distinct molecular mechanisms. In the case of 'extracting' CPEs, potency (measured in terms of porcine skin conductivity) and the capacity for irritation (determined from cell viability of human epidermal keratinocytes) are directly correlated, making it difficult to increase both effectiveness and safety. In the case of 'fluidizing' CPEs, however, no correlation was found between irritation and efficiency. Using this knowledge, the authors modified ten known CPEs, and found a significant number of fluidizers that were better than the original ones. (Proc. Natl. Acad. Sci. USA 102, 4688–4696, 2005) TM

Feline allergy relief

Although our understanding of the cascading reactions that lead to allergic reactions is advancing, treatments for people suffering from allergies remain rudimentary. Injections with low doses of antigens that can drag on for years are the current standard of care, often with variable success. Now an approach reported by researchers at the University of California, Los Angeles, and Virginia Commonwealth may one day provide enhanced allergy treatment. Their strategy involves the creation of chimeric antibody consisting of a truncated portion of the human Fc region fused with a cat allergen, Fel d1, which disrupts the allergic cascade when tested both in vitro and in vivo. Normally, multivalent antigens crosslink FcεRI receptors on basophiles and mast cells, leading to cascading phosphorylation and eventual histamine release. But these cells also contain a receptor, FcγRIIb, which when aggregated with FcεRI, disrupts the pathway. The chimeric antibody aggregates FcγRII with FcεRI, and thus prevents FcεRi-mediated phosphorylation. In vitro, the fusion protein blocked in a dose-dependent manner histamine release from mast cells taken from people allergic to cats, and in vivo, it prevented allergic reactions in both passively and actively sensitized mice. This potential therapeutic platform could be used with other allergens and might prove to be safer than antigen injections. (Nat. Med. 11, 446–449, 2005) LD

Simplicity in reverse

Dissecting the network structure of both normal and complex pathogenic phenotypes of higher eukaryotic and mammalian cells remains one of the key challenges for contemporary biologists. In an effort to develop new methods to reconstruct cellular networks from genome-wide data, a process called network reverse engineering, Califano and colleagues propose a simplified way for establishing links among coregulated genes. The authors apply a basic principle of data transmission theory, the 'data processing inequality' (DPI), to reduce the amount of information put into the reconstruction exercise. Previously, scientists have tried to systematically analyze all possible links among all genes, whether direct or mediated by other genes, in either simpler prokaryotic and eukaryotic regulatory networks, or in subnetworks of complex eukaryotes. By eliminating all those interactions whose coregulation is mediated by one or more intermediate steps and concentrating only on direct interactions between two genes, application of DPI to network reverse engineering results in more accurate network reconstructions than previous Bayesian approaches. Using these principles, the authors create an algorithm for the reconstruction of accurate cellular networks and apply it to reverse engineer the regulatory network of human B cells. The resulting analysis identifies MYC as one of the major regulatory hubs for determining cell fate. (Nat. Genet. 34, 382–390, 2005) GTO

Interfering with neurodegeneration

RNA interference (RNAi) has been shown to reduce symptoms of amyotrophic lateral sclerosis (ALS) in a mouse model of the disease, according to two reports in Nature Medicine. Familial forms of ALS have been linked to over 100 mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) through toxic gain-of-function mechanisms. Working with mice that overexpress one of these mutant genes (SOD1G93A) and develop an ALS-like condition, Raoul et al. and Ralph et al. found that small hairpin RNAs targeted to SOD1 slow disease onset and progression and extend the animals' survival. Raoul et al. delivered the small hairpin RNA (shRNA) by a lentiviral vector injected into the lumbar spinal cord, which allows transduction of neurons and glia, whereas Ralph et al. used a lentiviral vector that reaches motoneurons by retrograde transport after injection into muscle. In both papers, the shRNA was designed to silence the normal form of human SOD1. To avoid the potential negative consequences of this, Raoul et al. produced a construct that expresses both the shRNA and an RNAi-resistant form of the normal gene. (Nat. Med. 11, 423–428, 2005; Nat. Med. 11, 429–433, 2005) KA

Research Highlights written by Kathy Aschheim, Nadia Cervoni, Laura DeFrancesco, Teresa Moogan and Gaspar Taroncher-Oldenburg.

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Research Highlights. Nat Biotechnol 23, 437 (2005).

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