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The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.

The Dax-1 protein is an enigmatic nuclear receptor that lacks an expected DNA binding domain, yet functions as a potent corepressor of nuclear receptors. Here we report the structure of Dax-1 bound to one of its targets, liver receptor homolog 1 (LRH-1). Unexpectedly, Dax-1 binds to LRH-1 using a new module, a repressor helix built from a family conserved sequence motif, PCFXXLP. Mutations in this repressor helix that are linked with human endocrine disorders dissociate the complex and attenuate Dax-1 function. The structure of the Dax-1:LRH-1 complex provides the molecular mechanism for the function of Dax-1 as a potent transcriptional repressor.

The orphan nuclear receptors SF-1 and LRH-1 are constitutively active, but it remains uncertain whether their activation is hormone dependent. We report the crystal structure of the LRH-1 ligand binding domain to 2.4 A resolution and find the receptor to be a monomer that adopts an active conformation with a large but empty hydrophobic pocket. Adding bulky side chains into this pocket resulted in full or greater activity suggesting that, while LRH-1 could accommodate potential ligands, these are dispensable for basal activity. Constitutive LRH-1 activity appears to be conferred by a distinct structural element consisting of an extended helix 2 that provides an additional layer to the canonical LBD fold. Mutating the conserved arginine in helix 2 reduced LRH-1 receptor activity and coregulator recruitment, consistent with the partial loss-of-function phenotype exhibited by an analogous SF-1 human mutant. These findings illustrate an alternative structural strategy for nuclear receptor stabilization in the absence of ligand binding.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Here we show that maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. We propose that this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction.

The screening of large numbers of compounds or siRNAs is a mainstay of both academic and pharmaceutical research. Most screens test those interventions against a single biochemical or cellular output whereas recording multiple complementary outputs may be more biologically relevant. High throughput, multi-channel fluorescence microscopy permits multiple outputs to be quantified in specific cellular subcompartments. However, the number of distinct fluorescent outputs available remains limited. Here, we describe a cellular bar-code technology in which multiple cell-based assays are combined in one well after which each assay is distinguished by fluorescence microscopy. The technology uses the unique fluorescent properties of assay-specific markers comprised of distinct combinations of different 'red' fluorescent proteins sandwiched around a nuclear localization signal. The bar-code markers are excited by a common wavelength of light but distinguished ratiometrically by their differing relative fluorescence in two emission channels. Targeting the bar-code to cell nuclei enables individual cells expressing distinguishable markers to be readily separated by standard image analysis programs. We validated the method by showing that the unique responses of different cell-based assays to specific drugs are retained when three assays are co-plated and separated by the bar-code. Based upon those studies, we discuss a roadmap in which even more assays may be combined in a well. The ability to analyze multiple assays simultaneously will enable screens that better identify, characterize and distinguish hits according to multiple biologically or clinically relevant criteria. These capabilities also enable the re-creation of complex mixtures of cell types that is emerging as a central area of interest in many fields.

In order to determine the importance of benthic protozoa as consumers of bacteria, grazing rates have been measured by using monodispersed fluorescently labeled bacteria (FLB). However, high percentages of nongrazing benthic protists are reported in the literature. These are related to serious problems of the monodispersed FLB method. We describe a new method using 5-(4,6-dichlorotriazin-2-yl)-aminofluorescein (DTAF)-stained sediment to measure in situ bacterivory by benthic protists. This method is compared with the monodispersed FLB technique. Our estimates of benthic bacterivory range from 61 to 73 bacteria protist -1 h -1 and are about twofold higher than the results of the monodispersed FLB method. The number of nongrazing protists after incubation for 15 min with DTAF-stained sediment is in agreement with theoretical expectation. We also tested the relative affinity for FLB of protists and discuss the results with respect to a grazing model.

SummaryVertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members—mouse and human SF-1 and human LRH-1—which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

A total synthesis of the complex, bent aromatic ring-containing marine alkaloid haouamine A is achieved through a route in which every step (with the exception of the final deprotection) is performed on a gram-scale. This is accomplished through the development of a method for the dehydrogenation of cyclohexenones that allows for point-to-planar chirality transfer. This strategy makes it possible to program the desired atropisomeric outcome from a simple chiral cyclohexenone. By synthesizing atrop-haouamine A, this work has firmly established that natural haouamine exists as a single, nonequilibrating atropisomer. Finally, biological investigations demonstrate that the bent aromatic ring of this natural product is critical for anticancer activity against PC3 cells.