Localization and regulation of yeast aldehyde dehydrogenase Ald4p structures.

Previously, we identified yeast strains, namely SWORD, showing more robust Ald4p-GFP filament formation than the typical strains. Here, we report that Ald4p-GFP in SWORD strains favorably polymerize into gigantic structures in the cytoplasm, despite the enzyme being established as a mitochondrial resident. In addition, we have found that nocodazole, a microtubule destabilizer, has no effect on Ald4p high-order assembly, suggesting no direct association between microtubule dynamics and Ald4p structure formation.

Differential translation elongation directs protein synthesis in response to acute glucose deprivation in yeast.

Protein synthesis is energetically expensive and its rate is influenced by factors such as cell type and environment. Suppression of translation is a canonical response to stressful changes in the cellular environment. In particular, inhibition of the initiation step of translation has been highlighted as the key control step in stress-induced translational suppression as mechanisms that quickly suppress initiation are well-conserved.

An In Vitro Assembly System Identifies Roles for RNA Nucleation and ATP in Yeast Stress Granule Formation.

Stress granules (SGs) are condensates of mRNPs that form in response to stress. SGs arise by multivalent protein-protein, protein-RNA, and RNA-RNA interactions. However, the role of RNA-RNA interactions in SG assembly remains understudied.

Conserved metabolite regulation of stress granule assembly via AdoMet.

Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress.

PRPS polymerization influences lens fiber organization in zebrafish.

Background: The self-assembly of metabolic enzymes into filaments or foci highlights an intriguing mechanism for the regulation of metabolic activity. Recently, we identified the conserved polymerization of phosphoribosyl pyrophosphate synthetase (PRPS), which catalyzes the first step in purine nucleotide synthesis, in yeast and cultured mammalian cells. While previous work has revealed that loss of PRPS activity regulates retinal development in zebrafish, the extent to which PRPS filament formation affects tissue development remains unknown.

A quantitative screen for metabolic enzyme structures reveals patterns of assembly across the yeast metabolic network.

Despite the proliferation of proteins that can form filaments or phase-separated condensates, it remains unclear how this behavior is distributed over biological networks. We have found that 60 of the 440 yeast metabolic enzymes robustly form structures, including 10 that assemble within mitochondria. Additionally, the ability to assemble is enriched at branch points on several metabolic pathways.

Saccharomyces cerevisiae ASN1 and ASN2 are asparagine synthetase paralogs that have diverged in their ability to polymerize in response to nutrient stress.

Recent work has found that many metabolic enzymes have the ability to polymerize in response to metabolic changes or environmental stress. This ability to polymerize is well conserved for the few metabolic enzyme paralogs that have been studied in yeast. Here we describe the first set of paralogs, Asn1p and Asn2p, that have differential assembly behavior.

Cup regulates oskar mRNA stability during oogenesis.

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development.

Common regulatory control of CTP synthase enzyme activity and filament formation.

The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved.

Kinesin-73 is a processive motor that localizes to Rab5-containing organelles.

Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73.

A functional genomic screen in planarians identifies novel regulators of germ cell development.

Germ cells serve as intriguing examples of differentiated cells that retain the capacity to generate all cell types of an organism. Here we used functional genomic approaches in planarians to identify genes required for proper germ cell development. We conducted microarray analyses and in situ hybridization to discover and validate germ cell-enriched transcripts, and then used RNAi to screen for genes required for discrete stages of germ cell development.

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster.

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate-mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity.

Non-hinge-binding pyrazolo[1,5-a]pyrimidines as potent B-Raf kinase inhibitors.

As part of our research effort to discover B-Raf kinase inhibitors, we prepared a series of C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamides. X-ray crystallography studies revealed that one of the more potent inhibitors (10n) bound to B-Raf kinase without forming a hinge-binding hydrogen bond. With basic amine residues appended to C-3 aryl residues, cellular activity and solubility were enhanced over previously described compounds of this class.

Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay.

Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Ralpha1 and IL-4R.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection.

Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body.

The Balbiani body or mitochondrial cloud is a large distinctive organelle aggregate found in developing oocytes of many species, but its presence in the mouse has been controversial. Using confocal and electron microscopy, we report that a Balbiani body does arise in mouse neonatal germline cysts and oocytes of primordial follicles but disperses as follicles begin to grow. The mouse Balbiani body contains a core of Golgi elements surrounded by mitochondria and associated endoplasmic reticulum.

Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies.

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic "P bodies," including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways.

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition.

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies.

Efficient protein trafficking requires trailer hitch, a component of a ribonucleoprotein complex localized to the ER in Drosophila.

Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. We have identified Drosophila trailer hitch (tral) as a gene that is required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless.

Synthesis and structure-activity relationship of novel 6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2-thiones as progesterone receptor modulators leading to the potent and selective nonsteroidal progesterone receptor agonist tanaproget.

Tanaproget represents a potential first-in-class nonsteroidal PR agonist for contraception with improved safety and side effect profiles versus currently available steroidal oral contraceptives. Additional SAR, biological activity, and structural information from a tanaproget/hPR-LBD (hPR-LBD = human progesterone receptor ligand binding domain) cocrystal structure will also be presented.

Mechanisms of translational regulation in Drosophila.

Translational regulation plays an essential role in many phases of the Drosophila life cycle. During embryogenesis, specification of the developing body pattern requires co-ordination of the translation of oskar, gurken and nanos mRNAs with their subcellular localization. In addition, dosage compensation is controlled by Sex-lethal-mediated translational regulation while dFMR1 (the Drosophila homologue of the fragile X mental retardation protein) controls translation of various mRNAs which function in the nervous system.

Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz.

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes.