Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.ISSN:1097-2765ISSN:1097-416

Genetically directed production of recombinant, isosteric and non-hydrolyzable ubiquitin conjugates

We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl‐tRNA synthetase/tRNA(CUA) pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage‐specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages

Активность микрофлоры как показатель токсичности морских донных отложений шельфовой зоны Черного моря и Керченского пролива

Изучена потенциальная активность донной микрофлоры в местах утечки остатков химических токсикантов, затопленных в период Второй Мировой войны ХХ в. Отмечены особенности восстановления жизнедеятельности микрофлоры при различных уровнях загрязнения донных отложений мышьяком и хлорированными органическими сульфидами. Полученные результаты перспективно использовать при оценке экологического состояния донных отложений в загрязненных прибрежных акваториях

Fluorescence Lifetime Assays: A Smart Solution for Inhibitor Profiling on Protease Panels

We are monitoring enzyme mediated peptide modifications, such as proteolytic cleavage, using fluorescence lifetime as the readout parameter of choice. Fluorescence lifetime (FLT) is an intrinsic parameter of the fluorescence emission, and is thus more robust against interferences compared to conventional readouts such as absorption, luminescence, or fluorescence intensity. Having set out to replace our standard fluorescence intensity assays based on dual-label peptidic substrates, we arrived at a toolbox approach to assay development for endo- as well as exopeptidases. As a probe we selected a blue-fluorescent acridone dye with a relatively long lifetime, which is affected by the spatial proximity of either one of the two natural amino acids, tyrosine and tryptophan. Labeling a protease substrate with this probe and incorporating one of the quenching amino acids on the other side of the scissile bond enabled us to monitor protease activity in vitro by quantifying the FLT of the probe. To date we have developed more than 50 assays for proteases from all four classes

Fluorescence Lifetime Assays

The benefits of fluorescence lifetime- (FLT-) based technologies have been highlighted for over a decade but thus far they have failed to gain widespread acceptance. Significant improvements in assay reagents and availability of HTS-compatible readers, however, have now delivered a cost-effective, robust technology applicable to broad range of therapeutic targets. This article charts FLT development to a highly attractive tool for drug discovery

Fluorescence Lifetime Assays – Current Advances and Applications in Drug Discovery

Importance to the field Currently fluorescence lifetime-based biochemical assays are experiencing a new boost because of the availability of novel dyes and advances in instrumentation. The novel blue-fluorescent dyes with a significantly longer fluorescence lifetime (FLT) than probes used before and with defined mechanisms for fluorescence quenching enable a predictable development of much more robust assays. Fluorescence lifetime-based assays have been successfully introduced recently for finding and characterizing inhibitors of proteases and protein kinases, two mayor enzyme classes of high interest in drug discovery. Areas to be covered A comprehensive overview over the recent developments and the latest applications in the field of the fluorescence lifetime assay technology is provided. Current applications of FLT-based assays in drug discovery are reviewed. What the reader will gain The reader will gain a broad overview and a deeper understanding of the FLT-based assay technology that was added recently to the portfolio of assay methods established in drug discovery. Take home message The fluorescence lifetime technology offers an attractive alternative to existing methods for assaying proteases and protein kinases due to a straight-forward, generic, toolbox-based assay development using single-labeled substrates. Moreover, the technology enables robust identification of compound fluorescence artifacts

A fluorescence lifetime-based assay for protease inhibitor profiling on human kallikrein 7

Fluorescence lifetime is an intrinsic parameter describing the fluorescence process. Changes in the fluorophore's physicochemical environment can lead to changes in the fluorescence lifetime. When used as the readout in biological assays, it is thought to deliver superior results to conventional optical readouts. Hence it has the potential to replace readout technologies currently established in drug discovery such as absorption, luminescence or fluorescence intensity. Here we report the development of an activity assay for human kallikrein 7, a serine protease involved in skin diseases. As a probe, we have selected a blue-fluorescent acridone dye, featuring a remarkably long lifetime that can be quenched by either of the 2 natural amino acids, tyrosine and tryptophan. Incorporating this probe and 1 of the quenching amino acids on either side of the scissile bond of the substrate peptide enables us to monitor the enzymatic activity by quantifying the increase in the fluorescence lifetime signal. A systematic investigation of substrate structures has led to a homogenous, microplate-based, compound profiling assay that yields inhibitory constants down into the single-digit nanomolar range. This type of assay has now been added to our standard portfolio of screening techniques, and is routinely used for compound profiling

A Fluorescence Lifetime-Based Assay for Abelson Kinase

We present a novel homogenous in vitro assay format, and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. A single probe attached to a peptidic substrate responds with changes in its fluorescence lifetime, depending on whether or not a nearby tyrosine is phosphorylated. We utilize this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as antibodies. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay suitable to a wide variety of other tyrosine kinases

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing Time Correlated Single Photon Counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by Time Correlated Single Photon Counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second