Biology subjects

Ashok, Y.

Publications and source records attributed to Ashok, Y..

7 recordsLinked to original sources

bioRxiv2025

Dosa: A method to covalently barcode proteinsfor high throughput biochemistry

Deep mutational scanning couples a proteins activity to DNA sequencing for high throughput assessment of the effects of all single amino acid substitutions, but it largely uses indirect assays, like growth, as proxy for protein activity. Here, we covalently link variant proteins in vivo to an RNA barcode by fusing them to E. coli tRNA (m5U54) methyltransferase TrmA (E358Q), which forms a covalent bond with a tRNA stem-loop. Following cell lysis, variant proteins are separated in vitro according to their biochemical properties and identified by their barcodes. We use this method, Dosa, to analyze a large pool of FLAG epitope variants for binding to an anti-FLAG antibody, to profile the cleavage preferences of variants of enteropeptidase and human rhinovirus 3C protease, and to measure the solubility of several hundred A{beta}(1-42) variants. This method should be amenable to numerous biochemical assays with proteins produced in E. coli or mammalian cells.

genomics↗

bioRxiv2023

Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains

Ubiquitination is a complex and reversible protein post-translational modification in which the subsequent action of enzymes belonging to three different families, broadly referred to as E1, E2 and E3, results in the covalent linking of ubiquitin to a target protein. While this linkage is canonically an isopeptide bond between the C-terminus of ubiquitin and the lysine residue of the target protein, Ser, Thr, and Tyr can also be susceptible to ubiquitination through an oxyester bond. Once ubiquitinated, multiple units of ubiquitin can be attached to the initial ubiquitin thus extending it to a chain of ubiquitins. Ubiquitination regulates multiple cellular processes, but it is best known as a modification that targets proteins for proteasomal degradation following the formation poly-ubiquitin chains linked through lysine 48 or 63 of ubiquitin. Dysregulation of ubiquitination has been associated with multiple types of cancer and efforts have been carried out to develop technologies that lead to the identification of inhibitors of the enzymes involved in the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor auto-ubiquitination of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a robust signal window with a robust average Z factor of 0.76. From a validatory screening experiment we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. Additionally, we have expanded the system to study deubiquitinases such as USP28 that lead to reduction of FRET due to hydrolysis of fluorescent poly-Ub chains.

biochemistry↗

bioRxiv2023

DTX3L and USP28 fine-tune DNA double strand repair through mutual regulation of their protein levels

The DNA damage response (DDR) relies on a complex protein network to maintain genomic integrity, yet the interplay between post-translational modifiers remains poorly understood. Here, we uncover a novel regulatory axis between the E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 at DNA double-strand breaks (DSBs). Our results reveal a sophisticated feedback mechanism in which DTX3L ubiquitinates USP28, leading to its proteasomal degradation, while USP28 counteracts by deubiquitinating both itself and DTX3L. This cross-regulation fine-tunes DSB repair in multiple pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), single-strand annealing (SSA), and microhomology-mediated end joining (MMEJ). Strikingly, the detrimental effects of USP28 depletion on these repair pathways were rescued by concurrent DTX3L knockdown. Collectively, our work uncovers a novel layer of DDR regulation in which DTX3L and USP28s antagonistic activities calibrate cellular responses to genotoxic stress, thus identifying promising therapeutic targets to combat diseases associated with genomic instability. HighlightsO_LIDTX3L and USP28 physically interact and colocalize in cellular sub-compartments, with the N-terminal D1-D3 domains of DTX3L primarily mediating the interaction C_LIO_LIDTX3L ubiquitinates USP28 for degradation, while USP28 deubiquitinates itself and DTX3L, creating a sophisticated feedback mechanism. C_LIO_LIThe DTX3L-USP28 circuit influences levels of key proteins like HIF-1, p53, and c-MYC, suggesting broader impacts on cellular stress responses. C_LIO_LIDTX3L and USP28 cooperatively regulate multiple DSB repair pathways, including NHEJ, HR, SSA, and MMEJ, with USP28 depletion effects rescued by DTX3L silencing. C_LI

biochemistry↗

bioRxiv2022

benzothiazole scaffold as versatile nicotinamide mimic allowing nanomolar inhibition of different PARP enzymes

Here we report [1,2,4]triazolo[3,4-b]benzothiazole (TBT) as a new inhibitor scaffold, which competes with nicotinamide in the binding pocket of human poly- and mono-ADP-ribosylating enzymes. The binding mode was studied through analogs and their crystal structures with TNKS2, PARP2, PARP14 and PARP15. Based on the substitution pattern, we were able to identify The 3-amino derivatives 21 (OUL243) and 27 (OUL232), as inhibitors of mono-ARTs PARP7, PARP10, PARP11, PARP12, PARP14 and PARP15 at nM potencies, with compound 27 being the most potent PARP10 inhibitor described to date with an IC50 of 7.8 nM and the first PARP12 inhibitor ever reported. On the contrary, hydroxy derivative 16 (OUL245) inhibits poly-ARTs with a selectivity towards PARP2. The scaffold does not possess inherent cell toxicity and the inhibitors can enter cells and engage with the target protein. This, together with favorable ADME properties, demonstrates the potential of the TBT scaffold for future drug development efforts towards selective inhibitors against specific enzymes.

biochemistry↗

bioRxiv2021

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways like DNA damage response and infection, making them attractive targets for therapeutic intervention. DTX3L, an E3 ubiquitin ligase, forms a heterodimer with PARP9. The complex has ubiquitin ligase activity and also ADP-ribosylates the C-terminus of ubiquitin on Gly76. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. By using individually produced proteins, we have studied the interaction between DTX3L and PARP9. We identify that the D3 domain (230 - 510) of DTX3L mediates interaction with PARP9 with nanomolar affinity and an apparent 1:1 stoichiometry. Our results also suggest the formation of a higher molecular weight oligomer mediated by the N-terminus of DTX3L (1-200). Furthermore, we show that ADP-ribosylation of ubiquitin at Gly76 is a reversible modification that can be removed by several macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination in an inter-regulatory manner.

biochemistry↗

bioRxiv2019

FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized before in detail. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues when compared to Aerococcus viridans lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

biochemistry↗

bioRxiv2019

Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin

Targeted pathogen-selective approach to antibiotic development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5, 1:1:1:2:1) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates -subunits of the heterotrimeric Gi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify mg-levels of truncated but highly active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 {micro}M and 6.8 {micro}M) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gi in living human cells with a low micromolar IC50-value (2.4 {micro}M). NSC228155 and NSC29193 might prove useful hit compounds in targeted B. pertussis-selective drug development.

microbiology↗