Nuclear receptors (NRs) are major targets for drug discovery and have key roles in development and homeostasis as well as in many diseases such as obesity, diabetes, and cancer. NRs are ligand-dependent transcription factors that need to work in concert with so-called transcriptional coregulators, including corepressors and coactivators, to regulate transcription. Upon ligand binding, NRs undergo a conformational change, which alters their binding preference for coregulators. Short ␣-helical sequences in the coregulator proteins, LXXLL (in coactivators) or LXXXIXXXL (in corepressors), are essential for the NR-coregulator interactions. However, little is known on how specificity is dictated. To obtain a comprehensive overview of NR-coregulator interactions, we used a microarray approach based on interactions between NRs and peptides derived from known coregulators. Using the peroxisome proliferator-activated receptor ␥ (PPAR␥) as a model NR, we were able to generate ligand-specific interaction profiles (agonist rosiglitazone versus antagonist GW9662 versus selective PPAR␥ modulator telmisartan) and characterize NR mutants and isotypes (PPAR␣, -␤/␦, and -␥). Importantly, based on the NR-coregulator interaction profile, we were able to identify TRIP3 as a novel regulator of PPAR␥-mediated adipocyte differentiation. These findings indicate that NR-coregulator interaction profiling may be a useful tool for drug development and biological discovery. Molecular & Cellular Proteomics 8:2212-2226, 2009. Nuclear receptors (NRs)1 are ligand-inducible transcription factors involved in development and homeostasis that play key roles in many diseases, including diabetes, cancer, and obesity (1). NRs consist of several functional domains, which exhibit varying degrees of conservation among members of the receptor family. The poorly conserved N terminus contains the activation function 1 (AF-1) domain, the activity of which is often regulated by post-translational modifications. Centrally located is the DNA binding domain, which is highly conserved among species and between nuclear receptors. The ligand binding domain (LBD), which is also relatively well conserved in terms of primary amino acid sequence, mediates ligand binding, and contains the powerful ligand-dependent activation function (AF-2). LBD crystal structures have revealed a canonical fold consisting of 13 ␣-helices and a small four-stranded ␤-sheet (2). Upon ligand binding, the AF-2 helix (also referred to as helix 12) is stabilized in an active state (3). Depending on the conformation of the LBD and its modulation by ligand, NRs can recruit or release transcriptional coregulator proteins that perform all of the subsequent reactions needed to induce or repress transcription of target genes (4). Coregulators are often components of large multiprotein complexes that act in a sequential and/or combinatorial fashion to modify chromatin and to recruit basal transcription factors and RNA polymerase II (5). In general, the transcriptional coregulator family consists of coa...

Nuclear receptors (NRs) are ligand-inducible transcription factors that play critical roles in metazoan development, reproduction, and physiology and therefore are implicated in a broad range of pathologies. The transcriptional activity of NRs critically depends on their interaction(s) with transcriptional coregulator proteins, including coactivators and corepressors. Short leucine-rich peptide motifs in these proteins (LxxLL in coactivators and LxxxIxxxL in corepressors) are essential and sufficient for NR binding. With 350 different coregulator proteins identified to date and with many coregulators containing multiple interaction motifs, an enormous combinatorial potential is present for selective NR-mediated gene regulation. However, NR-coregulator interactions have often been determined experimentally on a one-to-one basis across diverse experimental conditions. In addition, NR-coregulator interactions are difficult to predict because the molecular determinants that govern specificity are not well established. Therefore, many biologically and clinically relevant NR-coregulator interactions may remain to be discovered. Here, we present a comprehensive overview of 3696 NR-coregulator interactions by systematically characterizing the binding of 24 nuclear receptors with 154 coregulator peptides. We identified unique ligand-dependent NR-coregulator interaction profiles for each NR, confirming many well-established NR-coregulator interactions. Hierarchical clustering based on the NR-coregulator interaction profiles largely recapitulates the classification of NR subfamilies based on the primary amino acid sequences of the ligand-binding domains, indicating that amino acid sequence is an important, although not the only, molecular determinant in directing and fine-tuning NR-coregulator interactions. This NR-coregulator peptide interactome provides an open data resource for future biological and clinical discovery as well as NR-based drug design.

A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.

A highly porous inorganic material (Anopore) was shown to be an effective support for culturing and imaging a wide range of microorganisms. An inert barrier grid was printed on the rigid surface of Anopore to create a "living chip" of 336 miniaturized compartments (200/cm 2 ) with broad applications in microbial culture.There is a pressing need in microbiology for the development of automated, miniaturized, and multiplexed growth formats that improve on the petri dish and the microtiter plate (1, 3). One approach to microbial culture has been to use flexible organic membranes as a support, permitting growth and imaging in situ (2, 10). We suggest in this work that an inorganic and rigid porous surface may be a better choice for many applications. Anopore is an inert ceramic, an aluminum oxide that is formed in sheets by a high-pressure and etching technique (4,6,8), creating a uniquely porous planar material; up to 50% of the volume is pores. Anopore has been identified as a superior surface for the imaging of microorganisms compared to flexible organic membranes (8, 9), in part due to its flatness and limited autofluorescence. The motivation for this work was to test the suitability of Anopore in the creation of versatile new growth formats or "living chips." Anopore strips (8 by 36 mm; 60 m thick; 0.2-m-diameter pores; 3 ϫ 10 9 pores cm Ϫ2 ) were a gift from PamGene International ('s-Hertogenbosch, The Netherlands). A latex solution (masking fluid 052; Royal Talens, The Netherlands) was applied to one surface in 1-mm-thick lines using a mapping pen and allowed to polymerize at room temperature for 20 min. The strips were then washed with distilled water and twice with 96% (vol/vol) ethanol and air dried. The grid formed a surface barrier 0.5 mm wide and 0.4 mm high that delineated eight culture areas of 7 by 4 mm. The polymerized latex was not strongly autofluorescent: illumination in the 515-to 730-nm range required exposures of Ͼ5 seconds to saturate the chargecoupled device camera. Anopore strips were placed on an appropriate nutrient agar base (5), inoculated on the upper surface at a density of 100 to 2,000 CFU/mm 2 , and then incubated. Microcolonies were stained by transfer of these strips right-side up to a microscope slide covered with a 1-mm-thick film of 1% (wt/vol) solidified low-melting-point agarose (Sigma, The Netherlands) containing 10 M Syto-9 dye plus 40 M propidium iodide (PI) or 5 M hexidium iodide (HI) for bacteria and 20 M Fun-1 for yeasts (7) (all dyes were from Invitrogen, The Netherlands). Staining was for 20 min at room temperature for bacteria and 30°C for yeasts. These procedures allowed staining of the organisms on the Anopore surface through the pores without disruption of the microcolonies. Strips were then imaged directly (without coverslip, immersion oil, or fixative) using an Olympus BX-41 fluorescence microscope equipped with U-MWIBA filters (excitation spectrum of 460 to 490 nm, dichroic mirror splitting at 505 nm, and an emission spectrum of 515 to 550 nm, used for Syto9 an...

◥Loss of the RAS GTPase-activating protein (RAS-GAP) NF1 drives aberrant activation of RAS/MEK/ERK signaling and other effector pathways in the majority of malignant peripheral nerve sheath tumors (MPNST). These dysregulated pathways represent potential targets for therapeutic intervention. However, studies of novel single agents including MEK inhibitors (MEKi) have demonstrated limited efficacy both preclinically and clinically, with little advancement in overall patient survival. By interrogation of kinome activity through an unbiased screen and targeted evaluation of the signaling response to MEK inhibition, we have identified global activation of upstream receptor tyrosine kinases (RTK) that converges on activation of RAS as a mechanism to limit sensitivity to MEK inhibition. As no direct inhibitors of pan-RAS were available, an inhibitor of the protein tyrosine phosphatase SHP2, a critical mediator of RAS signal transduction downstream of multiple RTK, represented an alternate strategy. The combination of MEKi plus SHP099 was superior to MEKi alone in models of NF1-MPNST, including those with acquired resistance to MEKi. Our findings have immediate translational implications and may inform future clinical trials for patients with MPNST harboring alterations in NF1.Significance: Combined inhibition of MEK and SHP2 is effective in models of NF1-MPNST, both those na€ ve to and those resistant to MEKi, as well as in the MPNST precursor lesion plexiform neurofibroma.