Synthesis and Screening of a DNA-Encoded Library of Non-Peptidic Macrocycles.

There is considerable interest in the development of libraries of non-peptidic macrocycles as a source of ligands for difficult targets. We report here the solid-phase synthesis of a DNA-encoded library of several hundred thousand thioether-linked macrocycles. The library was designed to be highly diverse with respect to backbone scaffold diversity and to minimize the number of amide N-H bonds, which compromise cell permeability.

High-Throughput Quality Control Assay for the Solid-Phase Synthesis of DNA-Encoded Libraries of Macrocycles*.

There is considerable interest in the development of libraries of scaffold-diverse macrocycles as a source of ligands for difficult targets, such as protein-protein interaction surfaces. A classic problem in the synthesis of high-quality macrocyclic libraries is that some linear precursors will cyclize efficiently while some will not, depending on their conformational preferences. We report here a powerful quality control method that can be employed to readily distinguish between scaffolds that do and do not cyclize efficiently during solid-phase synthesis of thioether macrocycles without the need for tedious liquid chromatography/mass spectrometry analysis.

Global analysis of gene expression mediated by OX1 orexin receptor signaling in a hypothalamic cell line.

The orexins and their cognate G-protein coupled receptors have been widely studied due to their associations with various behaviors and cellular processes. However, the detailed downstream signaling cascades that mediate these effects are not completely understood. We report the generation of a neuronal model cell line that stably expresses the OX1 orexin receptor (OX1) and an RNA-Seq analysis of changes in gene expression seen upon receptor activation.

Discovery of Phosphorylated Peripherin as a Major Humoral Autoantigen in Type 1 Diabetes Mellitus.

A major goal in understanding autoimmune diseases is to define the antigens that elicit a self-destructive immune response, but this is a difficult endeavor. In an effort to discover autoantigens associated with type 1 diabetes (T1D), we used epitope surrogate technology that screens combinatorial libraries of synthetic molecules for compounds that could recognize disease-linked autoantibodies and enrich them from serum. Autoantibodies from one patient revealed a highly phosphorylated form of peripherin, a neuroendocrine filament protein, as a candidate T1D antigen.

RNAi screen indicates widespread biological function for human natural antisense transcripts.

Natural antisense transcripts represent a class of regulatory RNA molecules, which are characterized by their complementary sequence to another RNA transcript. Extensive sequencing efforts suggest that natural antisense transcripts are prevalent throughout the mammalian genome; however, their biological significance has not been well defined. We performed a loss-of-function RNA interference (RNAi) screen, which targeted 797 evolutionary conserved antisense transcripts, and found evidence for a regulatory role for a number of natural antisense transcripts.

Supramolecular organization of the repetitive backbone unit of the Streptococcus pneumoniae pilus.

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus.

Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency.

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform.

Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts.

Successful protein expression, purification, and crystallization for challenging targets typically requires evaluation of a multitude of expression constructs. Often many iterations of truncations and point mutations are required to identify a suitable derivative for recombinant expression. Making and characterizing these variants is a significant barrier to success.

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution.

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1).

Crystal structure of the human TRPV2 channel ankyrin repeat domain.

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.