Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. Here we show that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, we show that piRNAs are Dicer independent and that their 3' end likely carries a 2'O-Methyl modification.

Diacylglycerol lipase-a (DAGL-a) is an intracellular, multidomain protein responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. [1] 2-AG is an endogenous signaling lipid that interacts with the cannabinoid CB1 and CB2 receptors. [2] Little is known about the regulation of its biosynthetic pathway and it is largely unclear to what extent 2-AG is responsible for distinct cannabinoid CB1 receptor mediated biological processes. Selective inhibitors of DAGL-a may contribute to a more fundamental understanding of the physiological role of 2-AG and may serve as potential drug candidates for the treatment of obesity and neurodegenerative diseases. [3] Currently, there are no selective inhibitors and activity-based probes available for the study of DAGL-a. [4] The identification of selective DAGL-a inhibitors is hampered by a lack of structural knowledge of the target, and a lack of assays that make use of endogenous DAGL-a activity in proteomes. No crystal structures are available and no homology models have been reported to aid hit identification and to guide optimization of the inhibitors. Determination of the selectivity of the inhibitors in native tissues is important because DAGL-a belongs to the serine hydrolase family, which contains more than 200 members with various physiological functions. [5] Fluorophosphonate (FP)based probes are routinely employed in competitive activitybased protein profiling (ABPP) experiments to determine the selectivity of serine hydrolase inhibitors in complex proteomes. DAGL-a, however, does not react with these activitybased probes. [6] Therefore, a new probe that can label native DAGL-a would be of value for studying the potency and selectivity of novel DAGL-a inhibitors in brain proteomes.Here we present a strategy that combines a knowledge-based in silico design approach and the development of a novel activity-based probe (ABP), based on the nonselective DAGL-a inhibitor tetrahydrolipstatin (THL; also known as Orlistat, a drug used for the treatment of obesity). This strategy resulted in the rapid identification of DAGL-a inhibitors with a new chemotype and high selectivity in the brain proteome.To identify novel DAGL-a inhibitors, we built a pharmacophore model based on THL using Discovery Studio Software Suite from Accelrys. Since THL can assume many different conformations, we searched the protein crystallographic database for crystal structures with a bioactive conformation for THL. A cocrystal structure of THL with fatty acid synthase (pdb-code: 2PX6) was identified (Figure 1 A) [7] that contains the same Ser-His-Asp catalytic triad and typical a/b hydrolase fold motif as DAGL-a. In this cocrystal structure, the nucleophilic Ser of the enzyme is covalently attached to the carbonyl moiety of the lactone. We reconstituted the ester to form the b-lactone to recover the active warhead of THL. After optimization of the geometry of the lactone, the resulting conformation was used to generate two pharmacophore models (Figure...

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian β-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian β-glucosidases (see picture). These probes offer new ways to study β-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

The spore-forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae. Herein, we present the isolation and structure elucidation of the antibacterial and antifungal products of this gene cluster, termed paenilamicins. The unique structures of the paenilamicins give deep insight into the underlying complex hybrid NRPS/PKS biosynthetic machinery. Bee larval co-infection assays reveal that the paenilamicins are employed by P. larvae in fighting ecological niche competitors and are not directly involved in killing the bee larvae. Their antibacterial and antifungal activities qualify the paenilamicins as attractive candidates for drug development.

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